WO2009018623A1

WO2009018623A1 - Molecular markers and methods related thereto

Info

Publication number: WO2009018623A1
Application number: PCT/AU2008/001152
Authority: WO
Inventors: Joanne Louise Dickinson; James M Stankovich; Russell James Thomson; Liesel M Fitzgerald; Briony Patterson; Andrea Polanowski; James Dowling Mckay; Simon James Foote; Stephen Quinn; Annette Banks; David A Mackey; Terence Dwyer
Original assignee: TASMANIA THROUGH MENZIES RESEARCH INSTITUTE, University of
Current assignee: TASMANIA THROUGH MENZIES RESEARCH INSTITUTE, University of
Priority date: 2007-08-09
Filing date: 2008-08-08
Publication date: 2009-02-12
Anticipated expiration: 2010-02-09

Abstract

A method of evaluating prostate cancer by determining nucleotides in the ITGA2 gene. A risk haplotype or set of risk alleles has been identified comprising SNPs set out in Table 6, Table 9 and Figure (5). In some embodiments, the method comprises identifying the SNPs, or alleles and distinguishing between risk and non-risk alleles. Primers and probes are described for evaluating prostate cancer in a subject and kits and arrays comprising these. Methods also include monitoring expression levels of the ITGA2 gene in a subject. Methods of treatment or prevention are described comprising modulating the level or activity of alpha2betal integrin polypeptide or a ligand or down stream effector thereof. Some methods also include testing the subject for a risk allele. Modulators of the level or activity of alpha2betal integrin polypeptide or a ligand or down stream effector thereof are proposed for use in treatment or prevention of prostate cancer. Methods of determining markers for prostate cancer are described.

Description

MOLECULAR MARKERS AND METHODS RELATED THERETO

FIELD

The specification relates inter alia to methods for evaluating prostate cancer based upon the identification of molecular markers comprising genetic or proteinaceous sequences indicative of the risk of developing prostate cancer. The present invention relates to a range of diagnostic methods and agents, screening methods and therapeutic methods and agents for the treatment or prophylaxis of prostate cancer.

BACKGROUND

Bibliographic details of the publications referred to in the subject specification are also collected at the end of the specification. The reference in this specification to any prior publication or information derived from it, or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Cancer is the second leading cause of death in the developed world. Apart from the suffering it causes to patients and their families it is also one of the most expensive diseases to treat. If the total costs are considered, the annual economic burden to society is expected to be in the order of US$200 to 500 billion by 2010.

In many Western countries, prostate cancer [MIM176807] is the most commonly diagnosed cancer after skin cancer (Parkin, Lancet. Oncol, 2(9):533-543, 2001; Pentyala et ah, Med. Oncol., i7(2):85-105, 2000). Familial aggregation of the disease indicates that it has a genetic component (Carter et ah, Cancer Surv., ii:5-13, 1991; Gronberg et al., Am. J. Epidemiol, 146(7):552-557, 1997; Schaid et al, Am. J. Hum. Genet., 62(6): 1425- 1438, 1998); however, there has been limited success in identifying prostate cancer susceptibility genes. While a plethora of genome-wide linkage studies have implicated a number of susceptibility loci (Camp et al., Prostate, <5J(4):365-74, 2005; Lange et ah, Prostate, 57(4):326-334, 2003; Lange et al, Hum. Genet., 119(4): 400-407, 2006; Schleutker et al, Prostate, 57(4):280-289, 2003; Smith et al, Science, 274(5291):1371- 1374, 1996; Tavtigian et al, Nat. Genet, 27(2): 172-180, 2001; Xu et al, Am. J. Hum. Genet., 77(2):219-229, 2005), until recently there has been no strong consensus between published studies (Easton et al, Prostate, 57(4) :261-269, 2003). Evidence presented by the International Consortium for Prostate Cancer Genetics (ICPCG) (Xu et al, 2005 (supra)) proposes five suggestive linkage regions supporting previously reported putative susceptibility loci (5q; (Camp et al, 2005 (supra); Maier et al, Eur. J. Hum. Genet., ii(3):352-360, 2005; Wiklund et al, Prostate, 57(4):290-297, 2003) 8p; (Maier et al, 2005 (supra); Xu et al, Am. J. Hum. Genet., <5P(2):341-350, 2001) 15q; (Lange et al., 2006 (supra); Maier et al, 2005 (supra); Gillanders et al, J. Natl. Cancer Inst., 96(16): 1240-1247, 2004) 17q; 9 (Lange et al, 2003 (supra); Gillanders et al, 2004 (supra)) and 22q; (Camp et al, 2005 (supra); Lange et al, 2003 (supra); Chang et al, Prostate, d¥(4):356-361, 2005). Many of these replication studies have been based on either expanded or refined subsets of the same populations (2q; (Goddard et al, Am. J. Hum. Genet., 68(5): 1197-1206, 2001; Suarez et al, Am. J. Hum. Genet., <56(3):933-944, 2000) 22q; (Camp et al, 2005 (supra); Xu et al, 2005 (supra)), thus studies providing further independent evidence for linkage at these five loci are still required. Most recently genome-wide association studies in large independent case control datasets have provided compelling evidence for two different loci on 8q24 (Gudmundsson et al, Nat. Genet., 39(5):631-637, 2007; Haiman et al, Nat. Genet., 3P(5):638-644, 2007; Yeager et al, Nat. Genet., 39(5):645-649, 2007), however no susceptibility genes have been identified for either locus. Subsequently, evidence for additional loci on chromosomes 2, 3, 6, 7, 8, 10, 11, 17 and X has been provided (Eeles et al, Nat Genet 2008; 40: 316-321; Gudmundsson et al, Nat Genet 2008; 40: 281-283.; Thomas et al, Nat Genet 2008; 40: 310-315). Whilst many of these SNPs are located in or close to known genes, a functional role for these SNPs in prostate cancer is yet to be demonstrated.

While complex diseases are probably influenced by a mixture of common and rare genetic variants, there is evidence that rare variants play a larger role in cancer than in other diseases (Risch, Cancer Epidemiol. Biomarkers Prev., iθ(7):733-741, 2001; Wang et al, Nat. Rev. Genet., <5(2):109-118, 2005). Until the advent of genome-wide resequencing, linkage analysis with multiplex families remains the most powerful method for detecting rare variants (Risch, 2001 (supra)). In addition, large multiplex pedigrees can be used to help address the problem of disease heterogeneity and are therefore potentially advantageous when studying a complex disease such as prostate cancer.

There is need for a greater understanding of the pathophysiology of prostate cancer and for new methods of diagnosing, preventing and treating prostate cancer.

SUMMARY

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps consisting of

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO: 1), <400>2 (SEQ ID NO: 2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

The present invention relates to methods for evaluating prostate cancer or other hyperproliferative cellular diseases in a subject, including evaluating a subject's likelihood of developing prostate cancer or responding to medication or evaluating a subject's risk of or susceptibility to developing different types or forms of prostate cancer.

In some embodiments, the methods further comprise processing the results of the evaluation to provide a diagnosis or prognosis of a condition. In some embodiments, the methods further comprise generating a report that includes information on the presence or absence of individual target polynucleotides in the test sample and/or a diagnosis or prognosis of a condition or other presentation of the results of the evaluation. Conveniently, the processing is performed by a programmable computer.

In one embodiment, the present invention concerns methods for evaluating prostate cancer based on the identification of a 14Mb haplotype on chromosome 5 at 5pl3-ql2 in eight patients in a large prostate cancer pedigree from a population that is genetically and environmentally homogeneous (See Figure 1). More particularly, within this region the

ITGA2 gene (identified as hg8, location chromosome 5: nucleotides 52320913 to

52426366) which encodes the c& subunit (ITGA2 polypeptide) of the c.2/31 integrin receptor has been identified herein as the susceptibility gene within this region. Thus, molecular markers of susceptibility or resistance to prostate cancer including metastatic prostate cancer are identified, for example, by analysing the ITGA2 gene or its expression products, including RNA or proteinaceous molecules, to identify polymorphisms or allelic variants segregating with the disease phenotype. The present invention extends to variants of the herein disclosed molecular markers which may be naturally occurring or synthetically or recombinantly produced. Further markers may be identified by methods know in the art such as by sequence comparison in silico, single strand conformation electrophoresis, sequencing, RNase cleavage, chemical cleavage mismatch, or amplicon melting analysis. m one particular embodiment, a risk haplotype for prostate cancer or prostate cancer metastasis has been more closely identified as residing within the 3' UTR of ITGA2 and within the region comprising the "3' UTR in/del" of exon 30 (SNP rs3212649 see Table 9 and Figure 5).

Accordingly, in one embodiment, a method is provided for identifying molecular markers of susceptibility or resistance to prostate cancer. In some embodiments, the method comprises identifying a polymorphism (variant) associated with prostate cancer comprising screening the ITGA2 gene or parts thereof from a subject for a polymorphism or variation which segregates with the herein described disease susceptibility haplotype (extending from marker D5S2506 to marker D5S664) or disease phenotype. In some embodiments, the polymorphism functions to modulate ITGA2 gene regulation. Molecular markers may thus be tested for their ability to modulate ITGA2 gene expression as illustrated for example in Example 10. In some embodiments the invention provides a method of identifying a polymorphism associated with prostate cancer comprising screening the ITGA2 gene or parts thereof from a subject for a polymorphism which segregates with one or more risk alleles in the ITGA2 gene, hi some embodiments, the method further comprises testing the polymorphic sequence for its ability to modulate ITGA2 gene expression relative to controls.

In an illustrative embodiment, once the 3' UTR in/del polymorphism was identified as conferring significant risk associated with prostate cancer, the region comprising the nucleotide sequence in proximity to the 3' UTR in/del (rs 3212649) was mapped and additional markers identified such as: rs35440530*; rsl900182; rs6898333; rs57674800; rs7725246; and rs6880055. Polymorphisms (variants) at rs6880055 and rs57674800 are identified as within miRNA target motifs and as shown in Example 10, at least the risk allele in rs57674800 is shown to modulate ITGA2 expression.

The polymorphism may be a single or multiple nucleotide polymorphism, including one or more nucleotide insertions, substitutions, deletions or inversions. Alternatively, the variation may be in the methylation or other modification status of the nucleotide sequence. Single and other small nucleotide polymorphisms (SNPs) are collected into databases together with reference information identifying their exact position in a particular genome. This reference information can be an "rs" (reference SNP) number, a chromosome nucleotide number, an exon nucleotide number, flanking sequence information or some other system or combination of systems as indicated, for example, in the website for The National Center for Biotechnology Information (NCBI). The polymorphisms set out in Table 6 and 9 and Figure 5 are identified as segregating with the herein described chromosome 5 haplotype and further polymorphisms are detected using the methods and information disclosed herein. As will be appreciated by the skilled artisan, it is not essential that all the at-risk polymorphisms identified herein are present in a subject to indicate that they are at risk for prostate cancer or metastasis. Functional polymorphisms are readily established based upon the information provided herein, hi some embodiments, a population of evolutionarily related subjects (such as subjects of northern European ancestry) will have functional at-risk polymorphisms in LD with nonfunctional polymorphisms and the non-functional polymorphisms are useful diagnostic markers in such populations, as well as the functional polymorphisms.

In other embodiments, the method comprises identifying variants in the ITGA2 polypeptide, which segregate with the disease phenotype. In other embodiments, the level of gene expression is evaluated, hi some embodiments, the subject is human.

The identification of a prostate cancer susceptibility gene and molecular markers therefore permits the development of kits and assays for evaluating whether or not a subject has prostate cancer or is likely to develop prostate cancer or a particular form of prostate cancer or metastases.

In some embodiments, the present invention contemplates a method of evaluating prostate cancer in a human subject said method comprising treating a sample from a subject to determine the level of expression of IGTA2 mRNA encoding the α2 subunit of 0.2/31 integral, wherein an elevated level of expression relative to controls is indicative of an increased risk of developing prostate cancer or of developing metastatic prostate cancer. In other embodiments, the specification provides a method of evaluating prostate cancer in a human subject said method comprising treating a sample from a subject to determine the level of expression of IGTA2 mRNA encoding the o2 subunit of oϋβl integrin, wherein an elevated level of expression relative to controls is indicative that the subject will respond well to prostate cancer treatment or prophylaxis comprising administration of an o2/31 integrin antagonist. As the skilled artisan will appreciate suitable methods include comprise RT-PCR or Northern analysis.

In another embodiment, a method is provided for evaluating prostate cancer in a subject comprising: (a) obtaining a sample of a nucleic acid from a subject; and (b) treating the sample to determine the identity of one or more nucleotides in ITGA2 gene (hg8 location chr5: 52320913 to 52426366). In some embodiments, (b) comprises treating the sample to determine the identity of a single selected nucleotide or a subset of selected nucleotides in ITGA2 gene. In some embodiments, the method comprises determining the identity of one or more nucleotides selected from the group of SNPs identified in Table 6, Table 9, or Figure 5 or polymorphisms identified as being in LD therewith. In another embodiment, the method comprises determining the identity of the one of more nucleotides in the 3¹UTR of the ITGA2 gene. The nucleotide sequence of the 3¹UTR is represented in Figure 5 (this sequence is also represented in NCBI NM002203.3 which covers the region 3609 to 7869. In some embodiments, the nucleotides are one or more of a subset of selected polymorphisms or markers from the 3'UTR of the ITGA2 gene. While a range of markers have been identified herein, further markers may readily be identified in the population using the methods and information disclosed herein and the invention extends to markers (polymorphisms or variants) identified as linked to the 3' UTR in/del marker or one or more of the polypmorphisms described herein in Table 6 and Table 9 and in Figure 5 within the 3' UTR risk haplotype identified herein. In some embodiments, the subset comprises a group of polymorphisms identified in Figure 5 being those polymorphisms denoted as one or more of the polymorphisms selected from the group consisting of (i) the AAC deletion at rs3212649; (ii) the G allele at rs6880055; (iii) the 25bp insertion rs57674800; (iv) the CAAA deletion at rs35440530*; (v) the C allele at rsl900182; (vi) the C allele at rs7725246; and (vii) the A allele at rs6898333 (see Table 9). As shown herein, the polymorphisms denoted as rs3212649, rs57674800 and/or rs688005 are particularly contemplated as useful markers.

In some embodiments, the selected nucleotide/s is the AAC deletion at rs3212649. In another embodiment, the selected nucleotide/s in the G allele at rs6880055. In another embodiment, the selected nucleotide/s is the 25bp insertion at rs57674800. In another embodiment, the selected nucleotide/s is the CAAA deletion at rs35440530*. In another embodiment, the selected nucleotide/s is the C allele at rsl900182. In another embodiment, the selected nucleotide/s is the C allele at rs7725246. In another embodiment, the selected nucleotide/s is the A allele at rs6898333. In some embodiments, the selected nucleotides are two or more polymorphisms selected from (i) to (vii) above. In some embodiments, the selected nucleotides are three or more polymorphisms selected from (i) to (vii) above. In some embodiments, the selected nucleotides are four or more polymorphisms selected from (i) to (vii) above. In some embodiments, the selected nucleotides are five or more polymorphisms selected from (i) to (vii) above. In some embodiments, the selected nucleotides are six or more polymorphisms selected from (i) to (vii) above. In some embodiments, the selected nucleotides are each of polymorphisms selected from (i) to (vii) above.

In some embodiments, the selected nucleotides are (i) and one or more of (ii) to (vii), two or more of (ii) to (vii), three or more of (ii) to (vii), four or more of (i) to (vii), five or more of (ii) to (vii), six or more of (ii) to (vii). In some embodiments, the selected nucleotides are (i) and (ii) or (i) and (iii) or (i) and (ii) and (iii).

In some embodiments, one or both alleles in a subject are evaluated. In some embodiments, one or both strands of complementary nucleic acids are evaluated. In these cases, the skilled person with appreciate that the nucleotide or its complement or analogous form (U for T) may be identified.

In a particular embodiment, the subject is human. In some embodiments, the subject is of northern european ancestry.

In another embodiment, a method is provided for evaluating prostate cancer in a subject comprising: (a) obtaining a sample of a nucleic acid from a subject; and (b) treating the sample to determine the identity of one or more polymorphisms in ITGA2 gene (hg8 location chr5: 52320913 to 52426366). In some embodiments, (b) comprises treating the sample to determine the identity of a single selected polymorphism or a subset of selected polymorphisms in ITGA2 gene. In some embodiments, the method comprises determining the identity of one or more polymorphisms selected from the group of SNPs identified in Table 6 or Table 9 or polymorphisms identified as being in linkage disequilibrium therewith. In other embodiments, the method comprises determining the identification of the one of more nucleotides selected from the group of polymorphisms identified in Figure 5 being those polymorphisms denoted as one or more of rs3212649; rs35440530*; rsl900182; rs6898333; rs57674800; rs7725246; rs6880055.

In another embodiment, a method is provided for screening for mutations (SNPs, polymorphisms) in the ITGA2 gene in a subject comprising: (a) obtaining a sample of a nucleic acid from a subject; and (b) treating the sample to determine the identity of one or more nucleotides in ITGA2 gene (hg8 location chr5: 52320913 to 52426366). In some embodiments, (b) comprises treating the sample to determine the identity of a single selected nucleotide or a subset of selected nucleotides in ITGA2 gene. In some embodiments, the method comprises determining the identity of one or more nucleotides selected from the group of SNPs identified in Table 6 or SNPs identified in Table 9 or Figure 5. In another embodiment, the method comprises determining the identification of the one of more nucleotides selected from the group of polymorphisms identified in Figure 5 being those polymorphisms denoted as one or more of rs3212649; rs35440530*; rsl900182; rs6898333; rs57674800; rs7725246; rs6880055.

In another embodiment, a method is provided for screening for molecular markers of prostate cancer susceptibility in a subject comprising: (a) obtaining a sample of a nucleic acid from a subject; and (b) treating the sample to determine the identity of one or more nucleotides in ITGA2 gene (hg8 location chr5: 52320913 to 52426366. SEQ ID NO: 1 provides the cDNA sequence taken from NCBI NM-002203/ gi/116295257). In some embodiments, (b) comprises treating the sample to determine the identity of a single selected nucleotide or a subset of selected nucleotides in ITGA2 gene. In some embodiments, the method comprises determining the identity of one or more nucleotides selected from the group of SNPs identified in Table 6. In another embodiment, the method comprises determining the identification of the one of more nucleotides selected from the group of polymorphisms in the 3' UTR of the ITGA2 gene identified in Figure 5 being those polymorphisms denoted as one or more of rs3212649; rs35440530*; rsl900182; rs6898333; rs57674800; rs7725246; rs6880055.

In some embodiments of the above-described methods, the subject has been diagnosed with prostate cancer. In other embodiments, the subject has not been diagnosed with prostate cancer, or has a family history of prostate cancer or is suspected of having or being likely to have prostate cancer because of one or more other diagnostic or prognostic criteria. In an illustrative embodiment, the subject is over a prescribed age when routine diagnostic testing for prostate cancer is carried out. hi some embodiments, the subject is of northern European ancestry. In accordance with these methods, a subject exhibiting a polymorphism (variant or molecular marker) associated with prostate cancer can be diagnosed as having an increased risk of developing prostate cancer or developing metastases. Conversely, a subject who does not exhibit a polymorphism or variant associated with prostate cancer has a decreased risk of developing prostate cancer or has a decreased risk of developing metastases. hi some embodiments, the method comprises investigating nucleic acid from a subject to determine the identity of one or more nucleotides in a part of the ITGA2 gene, hi some embodiments, the method comprises investigating nucleic acid from a subject to determine the identity of one or more nucleotides in the 3' UTR of the ITGA2 gene. In another embodiment the method comprises determining the identity of one or more nucleotides in one or more exons selected from Exon 7, Exon 8, Exon 27 and Exon 30. For the avoidance of doubt, the sequence of the ITGA2 gene includes reference to the signal sequence, mature peptide encoding sequence, intron/exon boundaries, intron and exon sequences and 3' and 5' untranslated regions, all of which are in the public domain or are naturally occurring variants or corrected versions thereof. In some embodiments, the method comprises determining the identity of one or more nucleotides in the 3' UTR OΪITGA2 gene selected from the group consisting of one or more of

(i) Deletion or presence of nucleotides AAC or its complement (GTT) in SNP rs 3212649 (hgl8, rs 3212649, clir 5: 52422659-52422661) identified as nucleotides 4161-4163 in Exon 30 of SEQ ID NO: 1 and underlined nucleotides #554-556 in Figure 5 and nucleotides 554-556 in SEQ ID NO: 3;

(ii) Deletion or presence of nucleotides CAAA (or its complement TTTG) in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52424712 in HapMap database, identified as rs35440530*, or as underlined nucleotides #2598- 2601 in Figure 5 and SEQ ID NO: 3;

(iii) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52424756 in HapMap database, identified as SNP rsl900182 or as underlined nucleotide #2651 in Figure 5 and SEQ ID NO: 3;

(iv) Nucleotide T(U), C, A, or G in Exon 30 (3¹UTR) of ITGA2 gene, landmark chr 5: 52425126 in HapMap database, identified as SNP rs6898333 or as underlined nucleotide #3021 in Figure 5 and SEQ ID NO: 3;

(v) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52425126 in HapMap database, identified as SNP rs6880055 or as underlined nucleotide #3080 in Figure 5 and SEQ ID NO: 3; (vi) Deletion or presence of nucleotides

TATATAAACAACTTTGTAGGACTAT (SEQ ID NO: 4) or its complement (ATAGTCCTACAAAGTTGTTTATATA SEQ ID NO: 5) in Exon 30 (3'UTR) of ITGA2 gene, chr 5, identified as SNP rs57674800 in NCBI database or as an insertion of 25 nucleotides between nucleotide #3680 and 3681 in SEQ ID NO: 3 or between nucleotide #3680 and 3681 in

Figure 5 (top strand) and SEQ ID NO: 3; and

(vii) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5:52426172 in HapMap database, identified as SNP rs7725246 or as underlined nucleotide #4067 in the Figure 5 or nucleotide #4067 in SEQ ID NO: 3.

In some embodiments, the method comprises determining the identity of one or more nucleotides in the ITGA2 gene selected from the group comprising one or more of:

(i) deletion or presence of nucleotides AAC or its complement (GTT) in SNP rs 3212649 (hgl8, rs 3212649, chr 5: 52422659-52422661) identified as nucleotides 4161-4163 in Exon 30 in SEQ ID NO: 1 or as underlined nucleotides 554-556 in Figure 5 (top strand); (ii) nucleotide T(U), C, A or G in SNP rs 1126643 (hgl8, rs 1126643, chr 5:

52383126) identified as nucleotide 902 in Exon 7 of SEQ ID NO: 1; (iii) nucleotide T(U), C, A or G in SNP rs 1062535 (hgl8, rs 1062535, chr 5: 52387170) identified as nucleotide 968 in Exon 8 of SEQ ID NO: 1; and

(iv) nucleotide T(U), C, A or G in SNP rs 2303122 (hgl8, rs 2303122, chr 5:

52415034) identified as nucleotide 3395 in Exon 27 of SEQ ID NO: 1.

SNP rs3212649 is also referred to in the subject specification as "3'UTR in/del". SNP rsl 126643 is also referred to as C807T in the specification. SNP rslO62535 is also referred to as G873A in the specification. SNP rs2303122 is also referred to as C3300T in the specification. Using the above numbering system relative to SEQ ID NO:1 the nucleotide change in the identified SNP is C902T in rsl 126643; G968A in rslO62535; and

C3395T in rs2303122. The nucleotide change or polymorphism identified in the SNP is from C in the predominant or 'wild type' sequence to T in the prostate cancer subject in, for example, "C3300T". Similarly for G to A in, for example "G968A" the "A" indicates the nucleotide/polymorphism found in the SNP which is therefore associated with prostate cancer. As know to those of skill in the art, the description of a nucleotide change from "C to T" or "G to A" indicates that the complementary strand is likely to have a complementary "G to A" or "C to T", respectively. A public database referring to a SNP may describe the polymorphism on either strand and the skilled artisan can readily check the sequence, and correct where necessary, and understand, and correct where necessary, the orientation of the sequence referenced in a public database.

(i) deletion or presence of nucleotides AAC or its complement in the 3' UTR of

Exon 30 (hgl8, rs 3212649, chr 5: 52422659-52422661); (ii) nucleotide T(U in mRNA), C, A or G at position 807 in Exon 7 (hgl8, rs

1126643, chr 5: 52383126); (iii) nucleotide T(U), C, A or G at position 873 in Exon 8 (hgl 8, rs 1062535, chr

5: 52387170); and (iv) nucleotide T(U), C, A or G at position 3300 in Exon 27 (hgl8, rs 2303122, chr 5: 52415034).

In another embodiment, the method comprises determining the identity of one or more nucleotides in the 3' UTR of ITGA2 gene selected from the group comprising one or more of:

(i) Deletion or presence of nucleotides AAC or its complement (TTG) in SNP rs 3212649 (hgl8, rs 3212649 see Table 9, chr 5: 52422659-52422661) identified as nucleotides 4161-4163 in Exon 30 of SEQ ID NO: 1 nucleotide 554-556 on SEQ ID NO: 3 and underlined nucleotides 554-556 in Figure 5;

(ii) Deletion or presence of nucleotides CAAA (or its complement TTTG) in rs35440530* of Exon 30 (3'UTR) (landmark chr 5: 52424712 in HapMap database), or as underlined nucleotides #2598-2601 in Figure 5. (iii) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52424756 in HapMap database, identified as SNP rsl900182 or nucleotide #2651 in Figure 5.

(iv) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52425126 in HapMap database, identified as SNP rs6898333 or as underlined nucleotide #3021 in Figure 5.

(v) Nucleotide T(U), C, A, or G in Exon 30 (3'UTR) of ITGA2 gene, landmark chr 5: 52425126 in HapMap database, identified as SNP rs6880055 or as underlined nucleotide #3080 in Figure 5.

(vi) Deletion or presence of nucleotides TATATAAACAACTTTGTAGG ACTAT (SEQ ID NO: 4) or its complement

(ATAGTCCTACAAAGTTGTTTATATA (SEQ ID NO:5) in Exon 30 (3'UTR) of ITGA2 gene, chr 5, identified as SNP rs57674800 in NCBI database or as an insertion of 25 nucleotides between nucleotide #3680 and 3681of Figure 5 and SEQ ID NO: 3. (vii) _Nucleotide T(U), C, A, or G in exon 30 (3¹UTR) of ITGA2 gene, landmark chr 5:52426172 in HapMap database, identified as SNP rs7725246 or as underlined nucleotide #4067 in the Figure 5.

As the skilled person will appreciate, one or both alleles may be evaluated in the subject by a number of techniques. One convenient method is allele specific PCR (Newton et al., Nucleic Acid Research, 17:2503-2516, 1989 that uses two primers which anneal to a target sequence adjacent to a site of the SNP: the 3' sequence is complementary to the SNP sequence and only primers which are perfectly complementary to the SNP sequence will be extended by DNA polymerase.

In another related aspect, the present invention provides a molecular marker comprising a sequence of nucleotides selected from or complementary to the sequence extending from marker D5S2506 to marker D5S664 for use in the diagnosis of prostate cancer, hi a preferred embodiment, the molecular marker comprises one or more variants of the ITGA2 gene which is significantly associated with prostate cancer, hi some embodiments, the molecular marker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 SNP/s or their complementary sequence identified in Table 6 or 1, 2, 3, 4, 5 or 6 SNPs as identified in Figure 5 and Table 9.

In another related aspect, an isolated nucleic acid molecule is provided comprising approximately 10 to 20 contiguous nucleotides of (or complementary to) the ITGA2 gene including the 3'UTR. hi some embodiments, the ITGA2 gene is identified as hg8 location chr5: nucleotides 52320913 to 52426366 or as set forth in SEQ ID NO: 1. In other embodiments, the nucleic acid comprises 10 to 20 nucleotides including or adjacent to nucleotides identified as a linked polymorphism, including one or more of those set out in Table 6 and/or Figure 5, or a functional variant thereof. In an illustrative embodiment, oligonucleotides are provided comprising approximately 10 to 20 contiguous nucleotides of (or complementary to) the ITGA2 gene (hg8 location chr5: 52320913 to 52426366, or SEQ ID NO: 1, or SEQ ID NO: 3) including or adjacent to the nucleotide positions wherein AAC (or its complement GTT) is absent at nucleotide 4161 to 4163 of SEQ ID NO:1 (hgl8, rs 3212649, chr 5: 52422659-52422661), or functional variants thereof. By "adjacent to" is meant in its broadest sense and includes sufficiently close to allow a fill-in reaction to take place as well close enough to allow amplification of polymorphic sequences positioned 3' to primers.

In another embodiment, the isolated nucleic acid molecule comprises approximately 10 to 20 contiguous nucleotides of the 3'UTR of ITGA2 and including or adjacent to one or more nucleotide/s selected from the following (i) the AAC deletion at rs3212649; (ii) the G allele at rs6880055; (iii) the 25bp insertion rs57674800; (iv) the CAAA deletion at rs35440530*; (v) the C allele at rsl900182; (vi) the C allele at rs7725246; and (vii) the A allele at rs6898333; or their complementary forms or non-risk forms, or a functional variant of said nucleic acid molecules. In some embodiments, the nucleic acid molecules are for use or when used in evaluating prostate cancer in a subject. Thus, sequences may be complementary to coding and/or non-coding nucleic acid sequences as appropriate in the present context.

In some embodiments, the isolated nucleic acid molecule comprises approximately 10 to 20 contiguous nucleotides of (or complementary to) a part of the gene encoding ITGA2 and including or adjacent to the SNP at rs 1126643 or its complementary form, or functional variants thereof for use in evaluating prostate cancer in a subject. Thus, sequences may be complementary to coding and/or non-coding nucleic acid sequences as appropriate in the present context.

In some embodiments, isolated nucleic acid molecules are provided comprising at least 10 to 20 contiguous nucleotides of (or complementary to) a corresponding part of the gene encoding ITGA2 including or adjacent to SNP rs 1062535 or its complementary form, or functional variants thereof for use in evaluating prostate cancer in a subject.

In some embodiments, isolated nucleic acid molecules are provided comprising at least 10 to 20 contiguous nucleotides of (or complementary to) the gene encoding ITGA2 (SEQ ID NO: 1, Accession No. GI: 116295257) including or adjacent to SNP rs2303122 or its complementary form, or functional variants thereof for use in evaluating prostate cancer in a subject.

In some embodiments, isolated nucleic acid molecules are provided comprising a contiguous sequence of nucleotides of a part of (a subsequence) or complementary to a part (subsequence) of the gene encoding ITGA2 polypeptide wherein the nucleic acid molecule includes or flanks (3¹ or 5') the polymorphism identified in SNP rs3212649 and/or SNP rs 1126643 and/or SNP rslO62535 and/or SNP rs2303122, and/or their complementary form/s wherein at least two SNPs associated with prostate cancer are present. In some embodiments, at least three or four SNPs (or their complements) are present. In one embodiment of the method cDNA is analysed. Here a nucleic acid molecule comprising rs 1124643 and rsl 06253 and/or their complementary sequences is provided wherein the molecule is less than 100 nucleotides in length is conveniently employed. hi some embodiments, the nucleic acid molecules are the complementary form of the nucleotide sequences set out in SEQ ID NO: 1, SEQ ID NO: 3 or in Figure 5. In other embodiments, the nucleic acid molecules are the coding sequence set out in SEQ ID NO: 1. hi other embodiments the nucleic acid molecules are variants of the sequences set out in SEQ ID NO: 1, SEQ ID NO; 3 or in Figure 5 or a subsequence thereof, including naturally occurring variants or synthetic forms, hi an illustrative embodiment, functional variants of nucleic acids, oligonucleotides, primers and probes may include nucleotide analogs, one to about five or more nucleotide substitutions, deletions, inversions or additions, or they may comprise detectable tags or reagents to facilitate screening.

Accordingly, the present invention contemplates in some embodiments, the use of functional variants of the naturally-occurring nucleotide sequences of parts or fragments of the ITGA2 gene, for example as nucleic acid molecules, oligonucleotides, primers or probes, hi some embodiments, the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more nucleotides, hi general, variants will display at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99 or 100% similarity to a reference ITGA2 sequence over an appropriate window of comparison. One example of a naturally occurring sequence is set forth in SEQ ID NO:1 which sets out the coding sequence of ITGA2. The 3¹UTR region of ITGA2 gene is set out in Figure 5, the top strand of which is set out in SEQ ID NO: 3 Desirably, variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99 or 100% sequence identity to a reference nucleotide sequence as, for example, set forth in SEQ ID NO: 1, SEQ ID NO: 3 or Figure 5. Moreover, sequences differing from the native or reference sequence by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides are contemplated. In other embodiments sequences differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 7O₅ 80, 90, 100 or more nucleotides but which retain one or more properties of the reference marker polynucleotide are contemplated. As the skilled person will appreciate, in some embodiments, variants in probe or primer sequences are made using known algorithms and protocols to improve, for example, hybridisation qualities, to reduce non- specific effects or to facilitate the use of mixed probe or primer reactions. The subject marker sequences also include polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to reference polynucleotide sequences (such as those disclosed in this specification, tables and figures) or the reverse complement thereof. In some embodiments, the isolated nucleic acid molecules or complements thereof are designed so that the nucleotide corresponding to the modified nucleotide to be detected is located at the 3' end of the molecule. m other embodiments, the present invention provides a nucleic acid probe (or primer) selected from the group consisting of: (a) a probe that hybridises under high stringency conditions to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2, said nucleic acid molecule having T or its complement A in SNP rs 1126643 but not to a corresponding nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having C or its complement G in SNP position rs 1126643;

(b) a probe that hybridises under high stringency conditions to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having A or its complement T in SNP rs 1062535 but not to a corresponding nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having G or its complement C in SNP rs

1062535;

(c) a probe that hybridises under high stringency conditions to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having G or its complement C in SNP rs 2303122 but not to a corresponding nucleic acid comprising a sequence selected from the gene encoding ITGA2 molecule having A or its complement T in SNP rs 2303122; and

(d) a probe that hybridises under high stringency conditions to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having AAC (or GTT) deleted in SNP rs3212649 but not to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 having AAC (or GTT) in SNP rs 3212649.

In some embodiments, the present invention provides an nucleic acid probe or primer for use in the evaluation of prostate cancer wherein the nucleic acid is selected from the group consisting of:

(a) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having AAC (or GTT) deleted in SNP rs3212649 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having AAC (or GTT) in SNP rs3212649;

(b) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having a 25 bp insertion (TATATAAACA ACTTTGTAGGACTAT (SEQ ID NO:4) (or its complement) in SNP rs57674800 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having no 25bp insertion

(TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4)) or its complement deleted in SNP rs57674800;

(c) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6880055 but does not hybridise to under high stringency conditions or amplify to a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6880055; (d) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having C (or G) in SNP rsl900182 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having T (or A) in SNP rsl900182;

(e) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6898333 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6898333;

(f) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) deleted in SNP rs35440530* but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) in SNP rs35440530*; and

(g) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the

3^? UTR of ITGA2 having C (or G) in SNP rs7725246 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs7725246.

As will be readily apparent, in other embodiments, the present invention provides a nucleic acid probe or primer for use in the evaluation of prostate cancer wherein the nucleic acid is selected from the group consisting of:

(a) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the

3' UTR of ITGA2 having AAC (or GTT) in SNP rs3212649 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3¹ UTR of ITGA2 having AAC (or GTT) deleted in SNP rs3212649;

(b) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the

3' UTR of ITGA2 having a 25 bp deletion (TATATAAACA ACTTTGTAGGACTAT (SEQ ID NO:4)) or its complement in SNP rs57674800 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having a 25bρ insertion

(TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4)) or its complement in SNP rs57674800;

(c) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6880055 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6880055;

(d) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the

3' UTR of ITGA2 having T (or A) in SNP rs 1900182 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having C (or G) in SNP rsl 900182; (e) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6898333 but does not hybridise to under high stringency conditions or amplify to a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6898333;

(f) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) in SNP rs35440530* but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) deleted in SNP rs35440530* ; and

(g) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs7725246 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having C (or G) in SNP rs7725246.

As will be described further herein, the subject probes or nucleic acid molecules are optionally detectably labelled and operated under appropriate assay conditions. In some embodiments, the invention comprises an array of nucleic acid molecules attached to a solid support, the array comprising a nucleic acid as defined hereinabove. In some embodiments, the array comprises the nucleic acid defined in part a) and/or b) and/or c) above.

In another embodiment, the present invention provides an array of nucleic acid molecules attached to a solid support, the array comprising an oligonucleotide that will hybridise to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 wherein nucleotides AAC (or TTG) in SNP rs 3212649 are absent, under conditions in which the oligonucleotide will substantially not hybridise to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 wherein nucleotides AAC (or GTT) are present in SNP rs 3212649.

In another embodiment, the present invention provides an array of nucleic acid molecules attached to a solid support, the array comprising an oligonucleotide that will hybridise to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 wherein nucleotides AAC (or TTG) in SNP rs 3212649 are present, under conditions in which the oligonucleotide will substantially not hybridise to a nucleic acid molecule comprising a sequence selected from the gene encoding ITGA2 wherein nucleotides AAC (or GTT) are absent in SNP rs 3212649.

In other embodiments, the array comprises an oligonucleotide that will distinguish between the present or absence of a particular allele or SNP within the 3' UTR risk haplotype region such as the AAC deletion at rs3212649. In another embodiment, the allele is the G allele at rs6880055. In another embodiment, the allele is the 25bp insertion at rs57674800. In another embodiment, the allele comprises the CAAA deletion at rs35440530*. In another embodiment, the allele comprises the C allele at rsl900182. In another embodiment, the allele/SNP is the C allele at rs7725246. In another embodiment, the allele SNP is the A allele at rs6898333. Thus the oligonucleotides are prepared to hybridise to one allele/SNP within the risk haplotype and not to hybridise to a non-risk allele/polymorphism.

In another embodiment, a linked polymorphism in the ITGA2 gene is associated with a change in the level of gene expression or a change in the composition or function of the expression product. For example, it is proposed herein that a 3'UTR in/del polymorphism at rs 3212649 is important in regulating ITGA2 mRNA. Additionally, rs 6880055 and rs57674800 are contained within predicted miRNA motifs and hence variant forms at this location will modulate gene expression. Thus, for example, a hypermorphic mutation is associated with elevated levels of gene expression or elevated activity of the expressed gene products. Conversely, a hypomorphic mutation is associated with a reduced level of gene expression or a reduced level of or modified gene product activity compared to a wild type form of the allele not associated with prostate cancer. Where the expression product is a proteinaceous molecule diagnosis is, in some embodiments, conducted at the level of protein or glycoprotein analysis rather than nucleic acid analysis. In a particularly convenient embodiment, antibodies or other antigen-binding molecules are used to distinguish between mutant and wild-type forms of the ITGA2 polypeptide.

The subject methods may be developed in kit for suitable for home clinic, veterinary or filed diagnostic purposes. The subject methods may also be automated and/or controlled by computer software for high throughput screening.

The methods further comprise; (b) obtaining amplification product data that are characteristic of any amplified nucleic acid sequences produced by nucleic acid amplification; and (c) processing the amplification product data to determine which, if any, of the target polynucleotides is present in the sample. Suitably, the step of processing comprises comparing the amplification data to predetermined data that are characteristic of reference amplification products, wherein one or more of the reference amplification products is characteristic of an individual target polynucleotide, to determine which, if any, of the target polynucleotides is present in the test sample. In some embodiments, the comparison provides information to determine for each amplified nucleic acid sequence the combination of oligonucleotides from the set which has amplified that nucleic acid sequence.

The amplification product data suitably comprise one or more parameter values for each amplified nucleic acid sequence. Similarly, the predetermined data suitably comprise one or more parameter values for each reference amplification product. Representative parameters include mass, size, charge, electrophoretic mobility, melting temperature, sequence, hybridisation characteristics or the presence or absence of a molecule (e.g., nucleic acid, protein, lipid, carbohydrate or inorganic molecule), metal, ion, or label. In another embodiment, kits are provided comprising the herein described computer-readable medium and an array as defined herein, for genotyping the ITGA2 gene.

In another embodiment, the present invention provides a computer-readable medium comprising a plurality of digitally encoded genotype correlations selected from the ITGA2 gene correlation in Table 3 or the Examples wherein each correlation of the plurality has a value representing prostate cancer risk.

In some embodiments, the present invention provides a kit for evaluating prostate cancer in a subject, said kit comprising at least one reagent that selectively detects the presence or absence of a variation (polymorphism) in the ITGA2 gene.

Further contemplated are methods for preventing, or treating prostate cancer, or subtypes of prostate cancer for example metastatic prostate cancer comprising administering an agent that ameliorates the functional affect of a mutation in the ITAG2 gene in a cell or subject or corrects or inhibits a prostate cancer-inducing phenotype in a cell or subject. In some embodiments, the agent modulates and in some embodiments down regulates ITGA2 activity and the activity of dλβ\ integrm receptor. In other embodiments, the polymorphisms increase ITGA2 gene expression. As shown here in one embodiment, the 3' UTR prostate cancer risk haplotype functions to modulate expression of the ITGA2 gene leading to altered expression of the α2/31 receptor on the surface of prostate cancer cells. In accordance with one aspect of the present invention, methods of treatment or prevention are provided wherein an agent that inhibits the function of the o2/31 receptor is particularly useful in preventing or treating prostate cancer. Agents known to bind to and inhibit the cHβ\ receptor include without limitation endorepellin and functional variants thereof. In some embodiments, subjects are tested as described herein for an ITGA2 at risk haplotype or allele prior to treatment.

Methods of screening for agents useful for treating or preventing prostate cancer are also provided, hi an illustrative embodiment, screens comprise assaying for agents that compete with endorepellin or other integrin binding agents for binding to oQβl integrins.

The present methods of treating or preventing prostate cancer in a subject comprise administering an agent which modulates the level or activity of ITGA2 gene, or the gene encoding the integrin odβl receptor precursor or ITGA2 polypeptide and cHβ\ integrin receptor activity or a ligand or down stream effector, hi some embodiments, an agent which modulates the level or activity of one of the genes encoding the dλβλ integrin receptor is contemplated for use herein, hi some embodiments, subjects are screened for the presence of an ITGA2 at risk haplotype prior to treatment, hi a preferred embodiment the at risk haplotype contains one or more of the variant alleles at the seven SNPs (polymorphisms) set out in Figure 5; rs3212649; rs35440530*; rsl900182; rs6898333; rs57674800; rs7725246; rs6880055. hi another embodiment, an ITGA2/ α2/51 integrin receptor modulator or ITGA2 or α2jδl integrin receptor -ligand modulator is contemplated for use in the treatment or prevention of prostate cancer.

The subject agents include antibodies, inhibitory molecules such as iRNA, antisense oligonucleotides, gene therapy molecules or peptides which are derived from or are variants of, or are developed from, the o&βl integrin receptor or a subunit thereof such as an o2 or βl precursor molecule, or their ligands or binding partners. Other agents include those selected from in silico screening, high throughput chemical screening, function based assays or structure-activity relationships. The subject agents are conveniently provided in a medicament form such as a pharmaceutical composition.

Further, polymorphisms in the at-risk haplotype-defmed herein are disclosed in Example 10 and form part of the invention described herein, facilitating diagnosis.

One preferred agent is endorepellin or its functional variants which bind to the o& subunit of the oQβl receptor and inhibit its functional activity (see for example Woodall B et al, Journal of Biological Chemistry, 2S3(4):2335-2343, 2008).

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagrammatic representation of the PcTas9 prostate cancer pedigree showing the segregating haplotype across 5pl3-ql2. Only cases and individuals providing genotyping information pertinent to the cases are presented due to pedigree size. Two cases are not represented here (6 - brother of 132; 13 - uncle of 1, 2 and 474). The affected status of "older" generations is unknown as the Tasmanian Cancer Registry only lists patients' records since 1978. Shaded boxed areas represent the shared haplotype. Individuals genotyped with the Affymetrix 1OK array are indicated by an asterisk (*). Individuals genotyped from paraffin embedded tissue are indicated by a cross (+). ^# denotes age at diagnosis, followed by Gleason score presented in brackets.

Figure 2 is a graphical representation of the results of the Affymetrix 1OK array genome- wide scan. An NPL score of 5.58 (suggestive linkage threshold) is indicated by a horizontal dashed line. Figure 3 is a diagrammatic representation of the 14Mb region of interest on chromosome 5. Panel A shows the location and relative size of prioritised genes for re- sequencing. Panel B shows an expanded view of the ITGA2 gene. Exons are represented as tall black rectangles. Short grey lines represent SNPs identified from the HapMap and SeattleSNPs databases that are in strong linkage disequilibrium (r²>0.72) with 3¹UTR in/del. The relative positions of the five exonic SNPs discussed in the text are shown.

Figure 4 provides the nucleotide sequence of SEQID NO: 1 and highlights the intra exon SNP positions in this cDNA sequence. SNP rs3212649 is nucleotides 4161-4163 in SEQ ID NO: 1; SNP rsll26643 is nucleotide 902 in SEQ TD NO: 1; SNP rslO62535 is nucleotide 968 in SEQ ID NO: 1; SNP rs2303122 is nucleotide 3395 in SEQ ID NO: 1. Figure 5 is a diagrammatic representation of the ITGA2 3' untranslated region showing the position of the polymorphisms of interest in the region of the 3' UTR in/del polymorphism comprising the risk haplotype. First line of DNA sequence displays reference DNA strand. Second line of DNA sequence displays alternative DNA sequence at each SNP. Third line of DNA sequence shows displays sequence with * under those bases altered at each polymorphism. Note that the "CAAA" deletion listed here occurs at the same DNA location as a "AAA" deletion listed as rs35440530 in the NCBI database. It is not known at present whether this represents an error in the NCBI database or two different polymorphisms occurring at this location. DNA Sequence Reference: NCBI Nucleotide Database Reference Sequence ID NM002203.3 covering the region 3609....7869. The top strand in Figure 5 is represented in SEQ ID NO: 3. The nucleotide sequence numbering between the top and bottom strands of Figure 5 diverges at rs57674800.

Figure 6 is a graphical representation showing that the 3' UTR risk haplotype functions to alter levels of the reporter transcript, and thus alters oQβl receptor levels on the prostate cell surface. Constructs comprising the 3' UTR of ITGA2 from at-risk and non-risk genotypes were generated in a luciferase reporter construct (comprising the pMIR-REPORT luciferase vector) and transfected into the human prostate cancer cell line, PC3. Luciferase reporter gene assays were performed to examine the influence of genotype of ITGA2 mRNA levels.

PC - Luciferase construct comprising the pMIR-REPORT luciferase vector containing approximately 4.2 kilobases of the ITGA2 3 'mistranslated region comprising the "3'UTR prostate cancer risk haplotype" defined as comprising the following: the AAC deletion at Rs32112649, CAAA deletion at rs35440530*, the 'C allele at rsl900182, the 'A' allele at rs6898333, the 'G' allele at rs6880055, the 25bp insertion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'C allele at rs7725246. Wt - Luciferase construct comprising the pMIR-REPORT luciferase vector containing approximately 4.2 kilobases of the ITGA2 3 'mistranslated region comprising the "3'UTR wild-type or non-risk haplotype" defined as comprising the following: the AAC insertion at rs32112649, the CAAA insertion at rs35440530*, the 'T' allele at rsl900182, the 'G' allele at rs6898333, the 'A' allele at rs6880055, the 25bp deletion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'A' allele at rs7725246.

Nve - control (blank transfected)

Figure 7 is a graphical representation showing that the 25bp insertion ar re57674800 from the risk haplotype functions to modulate levels of the reporter contract indicating that it also alters receptor levels on the prostate cell surface. Risk - Luciferase construct comprising the pMIR-REPORT luciferase vector (Ambion) plus approximately 4.2 kilobases of the ITGA2 3'unstranslated region comprising the "3'UTR prostate cancer risk haplotype" defined as comprising the following: the AAC deletion at Rs32112649, CAAA deletion at rs35440530*, the 'C allele at rsl900182, the 'A' allele at rs6898333, the 'G' allele at rs6880055, the 25bp insertion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'C allele at rs7725246.

Wt - Luciferase construct pMIR-REPORT luciferase vector (Ambion) plus approximately 4.2 kilobases of the ITGA2 3'unstranslated region comprising the "3'UTR wild-type or non-risk haplotype" defined as comprising the following: the AAC insertion at rs32112649, the CAAA insertion at rs35440530*, the 'T' allele at rsl900182, the 'G' allele at rs6898333, the 'A' allele at rs6880055, the 25bp deletion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'A' allele at rs7725246.

25bp - Luciferase construct comprising the pMIR-REPORT luciferase vector

(Ambion) plus approximately 4.2 kilobases of the ITGA2 3 'untranslated region comprising the "3'UTR wild-type or non-risk haplotype", however the 25bp insertion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800 from the risk haplotype has been inserted.

Vector - Luciferase construct comprising the pMIR-REPORT luciferase vector (Ambion) alone.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides a description of the SEQ E) NOs provided herein. Table 2 provides an amino acid sub-classification. Table 3 provides exemplary amino acid substitutions.

Table 4 provides a list of non-natural amino acids contemplated in the present invention.

Table 5 provides genotype information and evidence for identity-by-descent (IBD) haplotype sharing on chromosome 5 for the 25 cases in the family PCT as9. Table 6 provides information concerning the ITGA2 SNPs identified by re- sequencing four PcTas9 haplotype carriers and two controls- at all SNPs the haplotype carriers share an allele that does not occur in either control.

Table 7 tabulates the results of testing the 3'UTR 3 base pair insertion (I)/deletion (D), C807T and C-52T polymorphisms for significant differences in allele frequencies between cases and controls using the M_QLS statistic (Thornton et al, Am. J. Hum. Genet., 2007, m press).

Table 8 tabulates the genotypic odds ratios calculated for the 3'UTR insertion (I)/deletion (D) and C807T polymorphisms using logistic regression.

Table 9 sets out selected nucleotide sequences comprising herein identified polymorphisms of the 3'UTR ITGA2 risk haplotype.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention contemplates diagnostic methods for evaluating prostate cancer in a subject. The invention is predicated, in part, upon the herein described common haplotype in a prostate cancer pedigree and further, the finding of ITGA2 as a susceptibility gene within this haplotype. ITGA2 encodes the o2 subunit of the 02/31 integrin receptor which has been implicated to be associated with tumour progression and metastases in non-prostate forms of cancer.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "a molecule" means one molecule or more than one molecule.

Each embodiment described in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

Reference to "cancer" or "cancer cells" includes cells that exhibit abnormal growth and which tend to proliferate in an uncontrolled way, and, in some cases lead to tumors and/or metastases.

The term "gene" is used in its broadest sense and includes genomic nucleic acid sequence at a particular genetic locus or an extrachromosomal nucleic acid sequence, such as the sequence of gene in a plasmid, and cDNA corresponding to the exons of a gene. Reference herein to a "gene" is also taken to include:-

(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or

(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'- untranslated sequences of the gene.

Reference to parts of the ITGA2 gene include;

(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3¹- untranslated sequences of the gene. A gene may include a region of a genome that is transcribed but not translated.

Publicly accessible databases contain the nucleotide sequence for ITGA2 genes, which may be updated or corrected from time to time, hi some embodiments, for example, the presently disclosed polymorphisms are located in the 3' UTR of the ITGA2 gene, the nucleotide sequence for which is contained in the NCRI nucleotide database under Reference No. NM002203 region 3. This sequence is also set out in figure 5 and SEQ ID NO: 3. hi this context it should also be noted that the ITGA2 polypeptide, α2131, has various synonyms as follows: intergrin o2; very late activation protein 2 receptor, alpha-2 subunit; VLA2 receptor, alpha-2 subunit, VLAA2; CD496; platelet glycoprotein Ia/IIa; platelet receptor for collagen, platelet alloantigen Br(a); HPA-5.

By "determine the identity of one or more nucleotides" is meant the application of any of the broad range of available techniques for distinguishing between one or more nucleotide sequences and is in no way limited to sequencing or other direct comparisons between nucleotide sequences.

Any subject who could benefit from the present methods or compositions is encompassed. The term "subject" includes, without limitation, humans and non-human primates, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds and any other organism. The most preferred subject of the present invention is a human subject. A subject, regardless of whether it is a human or non-human organism may be referred to as a patient, individual, subject, animal, host or recipient.

In some embodiments, diagnosis or prognosis of prostate cancer in a subject requires analysis of the present molecular marker in genetic form. Determining the presence or absence of a polymorphism or variation in a ITGA2 gene may comprise determining a partial nucleotide sequence of the DNA from said subject, said partial nucleotide sequence indicating the presence or absence of said polymorphism or variation. It may further be preferred to perform a polymerase chain reaction with the DNA from said subject to determine the presence or absence of said polymorphism or variation. Such techniques are known to those skilled in the art (see Lewin B, Genes V, Oxford University Press, 1994). Test samples may be prepared by any suitable protocol and may include saliva, urine, serum and plasma by way of non-limiting example. It is proposed that the subject test may be part of test to genotype other polymorphisms in a sample from the subject. The degree of sample and nucleic acid purity required will vary with the sensitivity of particular methods employed. In this regard, reference may be made to PCR texts and papers on nucleic acid amplification and mutation detection and Laboratory Manuals for example Ausubel (Ed) Current Protocols in Molecular Biology, 5^th Edition, John Wiley & Sons, Inc, NY, 2002, and Sambrook, Molecular Cloning: A Laboratory Manual, 3^rd Edition, CSHLP, CSH, NY, 2001. Sample extracts of RNA may be prepared by any suitable protocol as for example described in Ausubel (Ed), 2002 {supra), Sambrook, 2001, {supra) and Chomczynski et al, (Anal. Biochem., 162:156, 1987, hereby incorporated by reference).

Genomic DNA or RNA or cDNA may be fragmented to facilitate the analysis. Particular fragments may be enriched and unwanted fragments removed. Nucleic acids may be amplified using any suitable nucleic acid amplification technique. Several template dependent nucleic acid amplification processes are available for amplification of nucleic acids. For example, the polymerase chain reaction method (PCR) as described by Mullis et al., (see U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; and European Patent Application Nos. 86302298.4, 86302299.2, and 87300203.4, and Methods Enzymol., 755:335-350, 1987), is one of the most prominent methods. PCR involves the use of a pair of specific oligonucleotides as primers for the two complementary strands of the double- stranded DNA containing the target sequence. The primers are chosen to hybridize at the ends of each of the complementary target strands, 3' of the target sequence. Template- dependent DNA synthesis, on one or both strands, can then be catalysed using a thermostable DNA polymerase in the presence of the appropriate reagents. A thermal cycling process is required to form specific hybrids prior to synthesis and then to denature the double stranded nucleic acid formed by synthesis. Repeating the cycling process geometrically amplifies the target sequence. The determination of a level of transcription products of a ITGA2 gene can be performed in a sample from a subject using Northern blots with probes specific for said gene. Another preferred method of measuring said level is by quantitative PCR with primer combinations which amplify said gene-specific sequences from cDNA obtained by reverse transcription of RNA extracted from a sample of a subject. Another preferred method for the analysis of transcription products is chip based microarray-technology. These techniques are known to those of ordinary skill in the art (see Sambrook and Russell, Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2000). Furthermore, the level and/or activity of a translation product of the gene can be detected using a Western blot analysis, an immunoassay, an enzyme activity assay, and/or binding assay. These assays can measure the amount of binding between said translation product and an anti-polypeptide antibody by the use of enzymatic, chromodynamic, radioactive, or luminescent labels which are attached to either the anti-polypeptide antibody or a secondary antibody which binds the anti-polypeptide antibody. In addition, other high affinity ligands may be used. Immunoassays which can be used include e. g. ELISAs, Western blots and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999).

Oligonucleotide primers for use in amplifying all or part of the ITGA2 gene are contemplated. A PCR method employing a reverse transcription step is also used with an RNA target using RNA-dependent DNA polymerase to create a DNA template. The PCR method has been coupled to RNA transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR method, using the double-stranded DNA as a template for the transcription of single- stranded RNA. (see, e.g., Murakawa et al, DNA, 7:287-295 1988).

If oligonucleotides are used to screen for molecular markers in ITGA2 gene, primers flanking exons are used in some embodiments.

There are, however, several non-PCR-based amplification methods that can be used for amplifying the marker regions. One type of non-PCR-based amplification method includes multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets (see, e.g., Burg et al, WO 89/01050; Gingeras et aL, WO 88/10315; Kacian and Fultz, EPO Application No. 89313154; Davey and Malek, EPO Application No. 88113948.9; Malek et al, WO91/02818 and U.S. Patent No. 5,130,238; Davey et al, U.S. Patent Nos. 5,409,818; 5,466,586; 5,554,517 and 6,063,603; Eberwine et al, U.S. Patent No. 5,514,545; Lin et al, U.S. Patent No. 6,197,554; and Kacian et al, U.S. Patent No. 5,888,779).

Another type of amplification method used to amplify and distinguish between allelic or mutated forms of the molecular marker uses a ligase chain reaction (LCR), as described, for example, in European Patent Publication No. 320,308. This method requires at least four separate oligonucleotides, two of which hybridize to the same nucleic acid template so their respective 3' and 5' ends are juxtaposed for ligation. The hybridized oligonucleotides are then ligated, forming a complementary strand on the nucleic acid template. The double-stranded nucleic acid is then denatured, and the third and fourth oligonucleotides are hybridized with the first and second oligonucleotides that were joined together. The third and fourth oligonucleotides are then ligated together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation.

Yet another amplification method is the Qβ replicase (Qβ) method, as described, for example, in PCT Publication Ser. No. WO 87/06270 and U.S. Patent No. 4,786,600, which uses a specific RNA probe which is capable of specific transcription by a replicase enzyme. The method requires the design and synthesis of RNA probes with replicase initiation sites.

Alternatively, palindromic probes can be used as described, for example, in EPO Publication Nos. 0427073A and 0427074A to form a hairpin with a nucleic acid target sequence. The probe contains a functional promoter located in the hairpin region from which RNA transcripts are produced.

There are also several versions of a strand displacement amplification method that uses one strand of DNA to displace same strand DNA sequences hybridized to their complementary DNA sequences to generate many copies of the target DNA sequences under isothermal conditions. Walker et al, Proc. Natl. Acad. Sci. U.S.A., §9:392-396, 1992; Walker et al, Nucl.

Acids Res., 20:1691-1696, 1992, European Patent Application No. EP 0 497272, and European Patent Application No. EP 0 500 224, describe an oligonucleotide-driven amplification method using a restriction endonuclease. The restriction endonuclease nicks the DNA/DNA complex to enable an extension reaction and, therefore, amplification.

Becker et al, EPO Application No. 88306717.5, describe an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex cleaved prior to the extension reaction and amplification.

Dattagupta et al. describe another version of the strand displacement amplification method, which employs a nucleic acid polymerase lacking 5' exonuclease activity and a set of oligonucleotide primers to carry out isothermal amplification without requiring exonuclease activity or restriction endonuclease activity (U.S. Patent No. 6,214,587).

Still another amplification method that can be used in an amplification of nucleic acid comprising all or part of the target mutant or wild-type molecular marker is rolling circle amplification. This method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermal reaction that replicates the target sequences exponentially (U.S. Patent No. 6,287,824 to Lizardi).

A related amplification method using a similar approach is termed ramification extension amplification (RAM), U.S. Patent No. 5,942,391 to Zhang et al. The RAM method involves hybridizing a target nucleic acid to several non-overlapping oligonucleotide probes that hybridize to adjacent regions in the target nucleic acid, the probes being referred to as capture/amplification probes and amplification probes, respectively, in the presence of paramagnetic beads coated with a ligand-binding moiety. Through the binding of a ligand attached to one end of the capture/amplification probe and the specific hybridization of portions of the probes to adjacent sequences in the target nucleic acid, a complex comprising the target nucleic acid, the probes and the paramagnetic beads is formed. The probes may then ligate together to form a contiguous ligated amplification sequence bound to the beads, which complex may be denatured to remove the target nucleic acid and unligated probes. Altematively, nucleic acid samples are not amplified prior to the analysis for polymorphisms.

The subject method comprises, in some embodiments, exposing the test sample comprising nucleic acid under pre-determined conditions which permit analysis of the nucleic acid to detect the presence or absence of a single polymorphism or multiple polymorphisms as determined herein to be associated with and increased or decreased risk of prostate cancer.

A large number of genetic polymorphism or mutation detection methods are available for use in the present invention. Many of these are conveniently adapted to high throughput analysis. Many of these are also standard reactions whose management and optimisation are routinely practiced by the skilled addressee. In an illustrative embodiment, sequencing, in situ hybridisation methods, mass spectroscopy (MALDI-MS, MALDI-TOF, MALDI-TOF PSD etc), gel or capillary electrophoresis, cleavage methods or homogeneous- or heterogeneous hybridization-based methods and particularly amplicon melting analysis and high-resolution melting analysis as described, for example, in Gundry et. al, Clinical Chemistry, 49:3, 396-406 (2003) are conveniently employed.

While oligonucleotide primers and probes are designed with their use in hybridisation and amplification reactions in mind, nevertheless the optimal conditions may be determined empirically using routine procedures and without undue experimentation. In this regard, reference may be made Laboratory Manuals for example Ausubel (Ed), 2002 (supra) and Sambrook, 2001, (supra).

The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotides and linkages between them are naturally occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule may vary depending on the particular application. An oligonucleotide is typically rather short in length, generally from about 10 to 30 nucleotides, but the term can refer to molecules of any length, although the term "polynucleotide" or "nucleic acid" is typically used for large oligonucleotides.

By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides. Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotides may be attached to the 5¹ end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotides or a stretch of non-complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer. Oligonucleotides (whether primers or probes) are readily synthesized by standard techniques. For example solid phase synthesis via phosphoramidite chemistry is described in US Patent Nos. 4,415,732 and 4,458,006. Other methods include phosphdi- and tri-ester methods. Oligonucleotides may be modified with detectable markers such as fluorscein, dyes, colloidal particles or equivalent molecules, or they may be modified with reactive functionalities to permit further labelling such as biotinylation or formation of sulfydryl derivatives. In one embodiment, primers are selected from the gene comprising sequences encoding the α2 subunit of the oQβl integrin receptor or its complement or substantially complementary forms thereof, hi some embodiments, the primers are selected from the sequence set forth in SEQ ID NO: 1 or its complement which provides the nucleotide sequence of the exons encoding the o2/31 integrin receptor. In other embodiments the primers have at least about 60% identity to SEQ ID NO: 1 over an appropriate window of comparison or hybridize to this sequence or its complement under stringent hybridization conditions. The particular, hybridisation conditions will depend upon art recognised parameters. hi another embodiment primers are selected or designed from the 3' UTR sequence set out in SEQ ID NO: 3 or a complementary form thereof or based upon one or more of the allelic sequences set out in Figure 5.

Methods for mutation detection are well known in the art starting with PCR as described in Saiki et al., Science, 250:1350-1354, 1985; Mullis et al, 1987 {supra); Saiki et al, PNAS USA, 55:6230-6234, 1989. These authors describe PCR and its application for genotyping polymorphisms using a reverse dot blot procedure using immobilized oligonucleotide probes. Other hybridisation based methods include GeneChip microarrays as described by Hacia et al., Genome Res., 5:1245-1258, 1998. Other probe types are respectively, dynamic allele-specific hybridization (DASH), peptide nucleic acid (PNA) and locked nucleic acid (LNA) probes, TaqMan and Molecular Beacons (see for example in relation to molecular beacons, Tyagi et al., Nature Biotechnol., 16:49-53, 1998). Methods for polymorphism analysis are reviewed by Landegren et al Genome Research 8:769-776, 1998 incorporated herein in its entirety by reference.

Other approaches for polymorphism detection feature allele-specific PCR or primer extension. Allele specific PCR (Newton et al., Nucleic Acid Research, 17:2503-2516, 1989) uses two primers which anneal to a target sequence adjacent to a site of a SNP: the 3' sequence is complementary to the SNP sequence and only primers which are perfectly complementary to the SNP sequence will be extended by DNA polymerase. Allele specific forward primers are used together with a common reverse primer to selectively amplify specific alleles. As polymerase dependent amplification requires a matched 3' base, the 3' portion of the allele specific primer is designed to only hybridise to one allele: the number of specific primers equals the number of different alleles. A target allele is detected based on the binding specificity of the specific primer whereas the common primer only serves to amplify a nucleic acid sequence if the specific primer hybridises to the specific allele. Primer extension methods (minisequencing) employ a single nucleotide which is complementary to the nucleotide at the site of the mutation to extend an adjacently hybridized primer. In some embodiments this approach is used to determine the presence of the SNPs set out in Table 6 or further polymorphisms linked thereto.

Strand Displacement Amplification (SDA and multiple SDA (MSDA)) described in US Patent No. 5,422,252 and Little et al, and Rolling Circle Amplification (RCA) described in Liu et al, J. Am. Chem. Soc, 775:1587-1594, 1996 and US Patent No. 5,854,033 and US Patent No. 6,642,034 provide important modifications and new applications. Nucleic Acid Sequence Based Amplification (NASBA) described in Sooknanan et al, Biotechniques, 77:1077-1080, 1994 and Q-Beta Replicase Amplification disclosed by Tyagi et al, (1996, supra)) also provide important methods as do the methods disclosed in Dong et al, Genome Research, 77:1418-1424, 2001. Various permutations on these techniques may also be employed as reviewed by Syvanen, Nature Genetics, 2:930- 940, 2001. m the present method, any useful combination of features of different reactions may be used to increase the discriminatory power of the method. The method can also conveniently employ ligation as an initial discriminatory step.

Oligonucleotide ligation assays are described in US Patent No. 4,883,750.

In some embodiments, detection process is based on a polymerase dependent amplification. One example of a polymerase dependent reaction is PCR, wherein an extension product synthesised from one oligonucleotide of an individual oligonucleotide pair, when separated from its complement, can serve as a template for synthesis of an extension product of the other oligonucleotide of the pair. Generally a thermostable DNA dependent or RNA dependent polymerase is employed however, the choice is strictly dependent upon the amplification method.

Alternatively, or in addition, the detection process is based on a ligase dependent amplification. In one example of this type, one oligonucleotide of an individual oligonucleotide pair hybridizes to a first target sequence, and the other oligonucleotide of the individual oligonucleotide pair hybridizes to a second target sequence that is adjacent to the first target sequence. The hybridized pair of oligonucleotides serve as substrates for ligation to produce a ligation product that comprises both oligonucleotides of the individual oligonucleotide pair. The particular ligase depends upon the method. For example T4DNA ligase is particularly efficient at ligating DNA ends hybridized to an RNA target. Nearly adjacent primers may be subjected to a fill-in reaction prior to ligation and amplification. The ligation product may be displaced to allow the production of further ligation products from the target sequence. In this case, a thermostable ligase (such as Ampligase) may be used. The ligation product may also be circularised and amplified by RCA by routine methods as described by Qi et al, in Nucleic Acids Res., 2P(22):el l6, 2001.

After the analysis step the products are assessed in order to generate product data which will be compared with predetermined data to determine whether a polymorphism in the ITGA2 gene is present in the test sample. The predetermined data comprise characteristics of reference products, hi some embodiments, the method comprises comparing the product data to predetermined data that are characteristic of reference products to determine whether a polymorphism is present in the test sample.

Homogeneous, solution phase amplification reactions are contemplated in which oligonucleotides are allowed to interrogate sample nucleic acids in solution and products are detected and distinguished using characteristics of the products detectable without further processing steps, hi some embodiments, the product is detectably modified to distinguish mutant sequences from non-mutant sequences. For example, generic dyes that intercalate into amplified DNA or RNA may be used to assess product formation in real time PCR assays. hi one embodiment, a method for distinguishing one or more alleles of the ITGA2 gene in a sample is provided comprising exposing a set of predefined oligonucleotide primers or probes as broadly described herein to genomic DNA prepared from a test sample under conditions that permit hybridisation of oligonucleotides to complementary target nucleic acid sequences and polymerase mediated amplification of distinct nucleic acids, hi some embodiments, the oligonucleotide primers are detectably modified at their 5' ends with additional non-target sequence nucleotides. Polymerase mediated PCR amplification is conveniently carried out with a thermostable DNA dependent polymerase. An initial denaturation step is carried out at about 95⁰C for approximately two minutes. Then ramped pre-cycle and cycle stages are carried out. The primers are designed to produce distinct amplification products of a distinguishable nature i.e., by number, size, length, mass, mobility, melting temperature, sequence, hybridisation or the presence of a detectable and/or distinguishable modification or attachment etc, as herein described, wherein one or more characteristics of any amplified product is assessed and are indicative of which, is any, of the target polynucleotides is present in a sample.

Optionally, real time assays are used to screen test samples. For example, real time PCR is carried out using a combined or co-ordinated thermocycler and fluorometer which can amplify specific nucleic acid sequences and measure their concentration simultaneously. The yield of amplified nucleic acid may be assessed using double stranded DNA intercalating dyes such as SYBR Green 1. In this way the existence of an amplified product may be detected, hi order to distinguish between amplified products the melting temperature or electrophoretic mobility of amplified products is conveniently assessed. Alternatively assay products may be assessed by their mobility in gels subjected to electrophoresis. In both cases, the addition of non-target sequence nucleotides to the 5' portion of oligonucleotides may facilitate distinguishing amplified products.

Alternatively, or in addition, various steps in the reactions may be carried out using, for example, solid supports, chromatography or electrophoresis to separate amplified products from unincorporated oligonucleotides. In some embodiments unhybridized oligonucleotides are removed from the sample prior to determining one or more characteristics of any amplified products, hi other embodiments the amplified nucleic acid sequences are immobilised prior to removal of unhybridized oligonucleotides or otherwise determining their characteristics.

Labelled oligonucleotide probes may also be used to measure specific amplified products in real-time PCR. A number of different assay formats are available in which the signal from the label, usually a fluorescent label or dye, is detected after extension of an oligonucleotide probe by a polymerase having 5'-3' exonuclease activity. For example, oligonucleotide probes are used bearing a fluorescent group at the 5' end and a quenching molecule at the 3' end. When both groups are close, the quencher quenches the signal from the fluorescence group. During the exponential phase of PCR, the exonuclease activity of the polymerase cleaves the fluorescence label which, freed of the quencher emits a detectable signal in a fluorometer. One problem with this approach is that the fluorescence molecule is not immobilised with the oligonucleotide. hi another approach, which is also useful in ligation based amplification assay, two oligonucleotides carry different or distinguishable fluorescent dyes which when brought together on adjacent target sequences produce a detectable fluorescence signal. These primer pairs are conveniently used in conjunction with multicolour detection systems, hi another version, oligonucleotide pairs are ligated to form a substrate for bacteriophage Φβ replicase.

Ligation mediated amplifications are conveniently employed in multiplexed screens in which all or part of one or more sets of oligonucleotides are screened against test samples comprising one or more than one target polynucleotide. hi one embodiment, the amplified products are immobilised by hybridising to complementary probes which are themselves attached or capable of attachment to a solid substrate. Such complementary probes are conveniently part of a high density nucleic acid array, hi another aspect, one or more of the promiscuous oligonucleotides is/are detectably modified and amplified nucleic acid sequences are immobilised by hybridising to complementary probes which are themselves attached to a solid substrate and form part of a high density nucleic acid array, hi some embodiments, the method comprises detection of the detectable modification in any amplified nucleic acid sequences and its position on the array. hi some embodiments, amplified nucleic acids are immobilised on a nucleic acid array and detection of a signal generated from a reporter or detectable molecule on the array is performed using an array reader. A detection system that can be used by a "chip reader" for example is described by Pirrung et al (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.

Preferably high discrimination hybridisation conditions are used. For example, reference may be made to Wallace et al, (Nucl. Acids Res., (5:3543, 1979) who describe conditions that differentiate the hybridisation of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et al, (Proc. Natl. Acid. Sci. U.S.A., §2:1585, 1985) who describe conditions for hybridisation of 11 to 20 base long oligonucleotides using 3M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al, (DNA Cell. Biol, P(7):527-534, 1990) describe hybridisation conditions that allow stringent hybridisation of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as "locked nucleic acids" (Christensen et al, Biochem. J., 354:481-484, 2001).

Generally, a hybridisation reaction can be performed in the presence of a hybridisation buffer that optionally includes a hybridisation optimising agent, such as an isostabilising agent, a denaturing agent and/or a renaturation accelerant. Examples of isostabilising agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts. Hybridisation accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Hybridisation buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM. Generally, hybridisation will be at temperatures normally used for hybridisation of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C. For probes longer than 14 nucleotides, 20° C to 50° C is preferred. For shorter probes, lower temperatures are preferred. A sample is incubated with oligonucleotides for a time sufficient to allow the desired level of hybridisation between the target sequences in the target polynucleotides and any complementary sequences. For example, the hybridisation may be carried out at about 45° C +/-10° C in formamide for 1- 2 days. After the hybrid-forming step the probes are, in some embodiments, washed to remove any unbound nucleic acid with a hybridisation buffer, which can typically comprise a hybridisation optimising agent in the same range of concentrations as for the hybridisation step. This washing step leaves only bound target polynucleotides. The hybridisation reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence.

Depending on the nature of a detectable molecule associated with an oligonucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a ρrobe:target polynucleotide/amplified product hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, as herein described, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means can be interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In a preferred embodiment, the set of probes is in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.

In some embodiments, a polymorphism in the gene encoding ITGA2 polypeptide causes an alteration in the expressed polypeptide product. In some embodiments, diagnostic agents are used to distinguish between the altered and the reference form of the polypeptide. Illustrative agents include aptamers or antibodies which are generated by the skilled artisan by routine methods without undue experimentation. hi further embodiments, functional assays of channel activity are employed to distinguish between allelic forms.

The terms "mutation", "polymorphism", "allele" or "allelic variant" are used interchangeably and in their broadest sense to encompass a detectable change (difference/alteration) in a genetic molecule relative to a reference molecule. Specifically, mutations, alleles or polymorphisms may include deletions, translocations, duplications, insertions, inversions, substitutions, point mutations missense mutations, non-sense mutations, splice-site mutations and the like of one or more nucleotides. The proteinaceous forms of the molecular marker may be made, for example, recombinantly or synthetically. The step of analyzing proteinaceous forms of the marker can be performed by any method known in the art or described herein. Methods for analyzing proteins are well known in the art, and include: sodium dodecyl sulphate- polyacrylamide gel electrophoresis ("SDS-PAGE"), isoelectric focusing, high pressure liquid chromatography, FPLC, thin layer chromatography, affinity chromatography, gel- filtration chromatography, ion exchange chromatography, and other standard biochemical analyses, immunodetection, protein sequencing, analysis with protein arrays, mass spectrometry, and the like. Thus, the invention includes those further analytical and/or quantification methods adopted to detect the molecular marker in proteinaceous form.

Methods for mass spectrometric protein analysis are routine in the art. Mass spectrometry methods have been used to quantify and/or identify proteins. (See, e.g., Li et ah, Tibtech., iS:151-160, 2000) hi further embodiments, the molecular marker in genetic or proteinaceous form is used to screen a plurality or library of molecules and compounds for specific binding partners for use in detection analyses, including, for example, aptamers, DNA molecules, RNA molecules, peptide nucleic acids, polypeptides, mimetics, antibodies, small, medium and large chemical molecules.

Aptamers are generated using the SELEX method to identify high affinity nucleic acid ligands to most proteins (described in US Patent Nos. 5,270,163, 5,580,737 and 5,567,588). hi one embodiment, aptamer pairs are selected which bind to the molecular marker at a site of a known mutation in the ITGA2 polypeptide, ligation of the adjacent aptamers is followed by selective amplification of the ligated aptamers with detection of products via a detectable marker. hi another embodiment, one or more antibodies comprising an antigen binding site that specifically binds the molecular marker are used for the detection of the marker in vitro or in vivo. Antibodies capable of specifically recognising the α subunit and variant forms thereof may be labelled with detectable markers and used in sandwich assays, antibody arrays and the like to enable analysis of the molecular marker.

An "antibody" includes monoclonal antibodies, polyclonal antibodies, minibodies, antibody fragments (e.g., Fab, Fab¹, F(ab').sub.2, Fv, sFv, Fc, etc.), chimeric antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity. The antibodies may be murine, rat, human, canine, camel, or any other origin (including humanized antibodies). Antibodies may be produced by a number of methods known in the art, including, for example, production by a hybridoma, recombinant production, or chemical synthesis. References include: Catty (Ed), {Antibodies: a practical approach, IRL Press, 1988-1989); Shepherd et al. (Eds), {Monoclonal antibodies: a practical approach, Oxford University Press, 2000); Harlow and Lane, (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1999); and Zanetti et al. (Eds), (The Antibodies, Harwood Academic Publishers, 1995). In one embodiments therapeutic antibody have the capacity for intracellular transmission and include antibodies such as shark antibodies, camelids and llama antibodies, scFv antibodies and intrabodies or nanobodies, e.g. scFv intrabodies and V_HH intrabodies. Such antigen binding agents can be made as described by Lui et al., 2007, BMC Biotechnol. 7:78; Harmsen & De Haard in Appl. Microbiol. Biotechnol. 2007 Nov;77(l):13-22; Tibary et al., Soc. Reprod. Fertil. Suppl. 2007, 64:297-313; Muyldermans, 2001, J. Biotechnol. 74:277-302; and references cited therein.

Immunogens and polypeptides may be produced, for example, by chemical synthesis. Methods for synthesizing polypeptides are well known in the art. In some embodiments, the polypeptide immunogen is synthesized with a terminal cysteine to facilitate coupling to haptens. Schedules of immunization of the host animal are established and use conventional techniques for antibody stimulation. Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells essentially using the general somatic cell hybridization technique of Kohler et al, Nature, 256:495-497, 1975. The technique involves fusing myeloma cells and lymphoid cells and culture of fused cells in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium in which only fused cells can flourish. Antibody producing hybridomas are cultured in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration.

Antibody agents which are allele specific or capable of distinguishing between polymorphic forms of the subject molecular marker may be sequenced and the polynucleotide sequence cloned into a vector for expression and storage. Alternatively, the polynucleotide sequence is used for genetic manipulation to "humanize" the antibody or to improve the affinity, avidity specificity, or other characteristics of the antibody. Neutralizing antibodies which act as integrin receptor antagonists are also used therapeutically in the treatment of prostate cancer.

In another alternative embodiment, antibodies may be made recombinantly by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et ah, Annu. Rev. Immunol., i2:433-455, 1994. Alternatively, phage display technology (McCafferty et ah, Nature, 345:552-553, 1990) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. For example, antibody phage display libraries may be panned in parallel against a large collection of synthetic polypeptides. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M 13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.

Immunoassays and flow cytometry sorting techniques such as fluorescence activated cell sorting (FACS) can also be employed to isolate antibodies which are useful in diagnosis or prognosis. Antigen-molecular marker complexes may be identified by any art conventional method, such as, without limitation: enzyme-linked or immunoassays (EIA or ELIZA), immunoelectrophoresis, immunodiffusion, flow cytometry, lateral flow assays, dipsticks, rapids and the like.

Genetically modified animals and cells or tissues therefrom are also contemplated. In some embodiments, an allele encoding an cd subunit which modulates receptor expression or function is expressed in genetically modified cells to screen for agents which override or ameliorate the activity of the said allele. Specifically, mutations in the α- subunit are introduced which elevate integrin receptor levels or activity in vivo and in in vitro cultures of modified cells. Conveniently, detection marker systems are also incorporated into the genetically modified animals using art conventional methods. Integrin receptor agonists or antagonists and variants, derivatives and analogs thereof are tested in the genetically modified animals described herein to select agents use in the treatment or prevention of prostate cancer.

The term "genetically modified" refers to changes at the genome level and refers herein to a cell or animal that contains within its genome a specific gene which has been altered. Alternations may be single base changes such as a point mutation or may comprise deletion of the entire gene such as by homologous recombination. Genetic modifications include alterations to regulatory regions, insertions of further copies of endogenous or heterologous genes, insertions or substitutions with heterologous genes or genetic regions etc. Alterations include, therefore, single of multiple nucleic acid insertions, deletions, substitutions or combinations thereof. A genetically modified animal, cell or tissue includes animals or cells from a transgenic animal, a "knock in" or knock out" animal, conditional variants or other mutants or cells or animals susceptible to co- suppression, gene silencing or induction of RNAi. In some embodiments, genetic sequences comprising the 3'UTR region of ITGA2 is genetically modified.

Conveniently, targeting constructs are initially used to generate the modified genetic sequences in the cell or organism. Targeting constructs generally but not exclusively modify a target sequence by homologous recombination. Alternatively, a modified genetic sequence may be introduced using artificial chromosomes or viral vectors. Targeting or other constructs are produced and introduced into target cells using methods well known in the art which are described in molecular biology laboratory manuals such as, for example, in Sambrook, 2001; Ausubel (Ed), 2002 (supra). Targeting constructs may be introduced into cells by any method such as electroporation, viral mediated transfer or microinjection. Selection markers are generally employed to initially identify cells which have successfully incorporated the targeting construct.

Genetically modified organisms are generated using techniques well known in the art such as described in Hogan et a!., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbour Laboratory Press, CSH NY, 1986; Mansour et ah, Nature, 55(5:348-352, 1988; Pickert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press, San Diego, CA₅ 1994. Stem cells including embryonic stem cells (ES cells) are introduced into the embryo of a recipient organism at the blastocyst stage of development. There they are capable of integration into the inner cell mass where they develop and contribute to the germ line of the recipient organism. ES cells are conveniently obtained from pre-implantation embryos maintained in vitro (Robertson et ah, Nature, 322:445-448, 1986). Once correct targeting has been verified, modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism. Alternatively, modified cells are allowed to aggregate with dissociated embryonic cells to form aggregation chimera. The chimeric organism is then implanted into a suitable female foster organism and the embryo allowed to develop to term. Chimeric progeny are bred to obtain offspring in which the genome of each cell contains the nucleotide sequences conferred by the targeting construct. Genetically modified organism may comprise a heterozygous modification or alternatively both alleles may be affected.

Another aspect of the present invention provides cells or animal comprising one, two or more genes or regions which are modified. For example, the genetically modified cells or animals may further comprise a gene capable of functioning as a marker or reporter for detection of modified cells. Alternatively, the instant animals may be bred with other transgenic or mutant non-human animals to provide progeny some of which exhibit one or both traits or a modified trait/s. Chimeric animals are also contemplated.

A reporter molecule is conveniently encoded by a reporter expression cassette or reporter construct. The reporter construct can be brought under the control of, for example, the ITGA2 gene regulatory elements, particularly those regulatory elements which modulate expression of the gene in particular cells.

A "marker gene" imparts a distinct phenotype to cells expressing the marker gene such as resistance to antibiotic, radiation or heat. By "reporter" is meant any molecule, protein or polypeptide which is typically encoded by a reporter gene and measured in a reporter assay. Reporters provide a detectable signal which permit an understanding of the activity of genetic sequences. A reporter protein should be distinguishable from other proteins and ideally, readily quantified. The reactivity between an epitope and an antibody determined thereby may readily be employed optionally together with second or further antibodies. Common reporter proteins include luciferase, chloramphenicol transferase (CAT), Beta- galactosidase (B-gal), or fluorescent proteins such as green fluorescent proteins (GFP). Reference herein to GFP is meant to encompass any fluorescent or light-emitting protein including those derived from jelly fish or other organisms and all homologues, derivatives, analogues including colour variants such as DSRed, HcRed, Clontech; or hrGFP, Stratagene). Preferably said reporter expression cassette encodes a fluorescent or other light emitting GFP. GFP reporters are readily detectable in live cells and are particularly useful and preferred in cell sorting applications.

The present invention provides methods of screening for agonists or antagonists comprising contacting the cell expressing the subject mutant form of ITGA2 with an agent and assaying for (i) the presence of a complex between the ITGA2 polypeptide and an agent or (ii) for the presence of complex between the polypeptide and a ligand or other binding molecule, by methods well known in the art. In such competitive binding assays the ITGA2 polypeptide or the ligand is labelled in order to assess the activity of the agent.

In one embodiment, the present invention also provides methods for screening for antagonists of integrin receptor activity, comprising exposing a variant ITGA2 polypeptide to an agent and assaying for:-

(i) the presence of a complex between the agent and the ITGA2 polypeptide; or (ii) a change in the interaction between the ITGA2 polypeptide and a ligand, binding partner; or (iii) a change in the level of an indicator of the activity of the ITGA2 polypeptide.

The ability of an agent to modulate the activity of a target molecule may be tested in in vitro assays. Target molecule may be expressed recombinantly or occur naturally or be up regulated in cells or cell lines which are useful in in vitro screens for agents.

Natural products, combinatorial synthetic/peptide, polypeptide or protein libraries or phage display technologies are all available in the art for screening for modulatory agents. A huge choice of high throughput screening methods are also available. Natural products include those from coral, soil, plant or the ocean or antarctic environments. Examples of suitable methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261: 1303; Carrellet al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al., 1994, J. Med. Chem. 37:1233. Thus, agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145; U.S. 5,738,996; and U.S. 5,807,683). Libraries of compounds may be presented, for example, in solution (e.g. Houghten, 1992, Bio/Techniques 13: 412-421), or on beads (Lam, 1991, Nature354: 82-84), chips (Fodor, 1993, Nature 364: 555-556), bacteria (US 5,223,409), spores (US 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. , 1992, Proc. Natl. Acad. Sci. USA 89:1865- 1869) or phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249: 404-406; Cwirla et a., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991,1 MoI. Biol. 222: 301-310).

Suitable o2/31 integrin antagonists bind to the receptor or a receptor ligand and down-regulate receptor activity. Antagonists include soluble forms of the receptor or variants forms or receptor ligands that bind to the receptor but fail to initiate receptor activity. One example of an agent that binds to the alpha receptor is endorepellin (see for example Woodall et al, 2008.

Accordingly, in one embodiment, the present invention provides a method of treating or preventing prostate cancer or prostate cancer metastasis in a subject comprising administering an agent which modulates the level or activity of ITGA2 gene or ITGA2 polypeptide (integrin alpha 2, o2/31 integrin) or a ligand or down stream effector. In some embodiments, an α2/31 integrin modulator or an oQβl integrin-ligand modulator for use in the treatment or prevention of prostate cancer, hi other embodiments, the modulator is an antibody that binds to oQβl integrin on prostate cells, hi some embodiments, the invention comprises an α2/31 integrin antagonist or an 02/31 integrin-ligand antagonist for use in the treatment or prevention of prostate cancer or prostate cancer metastasis. In other embodiments, the invention is an o2jSl integrin antagonist for use in the prevention of prostate cancer metastasis. In some embodiments, the antagonist is endorepellin or a functional variant thereof, hi some embodiments, the method further comprises evaluating the subject for prostate cancer according to the method of any one of claims 1 to 12 and wherein a subject exhibiting the risk genotype is treated with an o&βl integrin modulator. In some embodiments, the agent is an o&βl integrin antagonist. In other embodiments, the agent is an o2/31 integrin agonist, hi some embodiments, the modulator or antagonist is endorepellin or a functional variant thereof.

Another example of an agonist or antagonist agent is a protein, polypeptide or peptide or a derivative, analog or variant thereof or an antibody. In some embodiments the agent is derived from a polypeptide comprising the α-subunit of the o&βl integrin receptor, or parts or homologs, derivatives or variants thereof. Another example of an agent is a genetic molecule encoding ITGA2 polypeptide or a derivative thereof. The terms "genetic molecule" "nucleic acids" "nucleotide" "oligonucleotide" and "polynucleotide" include RNA, DNA, cDNA, genomic DNA, synthetic forms, mixed polymers, sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatised nucleotide bases. The term typically refers to oligonucleotides greater than 30 nucleotides in length.

In one embodiment, the agents are isolated by which is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide", as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment.

The term "variant" is generally used in its broadest context and includes functional or non-functional fragments, parts, derivatives, analogs and the like. The terms polypeptide, protein and peptide are also used interchangeably herein.

A "part" or "subsequence" in peptide form may be as small as an epitope comprising less than 5 amino acids or as large as several hundred kilodaltons. The length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues. A "part" or "subsequence" of a nucleic acid molecule is defined a having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600, 1000 nucleotides or nucleic acid molecules having any number of nucleotides within these values. In some embodiments, parts have about 10 to 600, 20 to 400, 20 to 300, or 20 to 200 bp.

Variants of ITGA2 polypeptide or ITGA2 polypeptide ligants such as perlecan and parts thereof are contemplated for use in the presently described therapeutic methods. "Variant" polypeptides include proteins derived from the native or "wild-type" protein which does not facilitate the development of prostate cancer. Variant protein or peptides comprise deletions (so-called truncation) or additions of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention include those which are biologically active, that is, they continue to possess or possess at least one biological activity relative to the native protein such as ligand binding or modified ligand binding. However, they include molecules which possess only some of the biological activities of the reference molecule. Alternatively, the variant polypeptides are not biologically functionally active but are useful in the diagnostic or prognostic methods of the present invention. Variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of the present molecular marker polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a marker polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. The isolated, recombinant or synthetic marker polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the encoding nucleic acid sequence. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 52:488-492, 1985), Kunkel et al., (Methods in Enzymol., ./54:367-382, 1987), U.S. Patent No. 4,873,192, Watson et al. ^Molecular Biology of the Gene", Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do or do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al, (Natl. Biomed. Res. Found, 5:345-358, 1978).

Variant polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to the reference amino acid sequence. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine). Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterises certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al, 1978 {supra); and by Gonnet et al, Science, 256(5062): 1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.

Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 2. Conservative amino acid substitution also includes groupings based on side chains.

For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.

For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional ITGA2 polypeptide or an ITGA2 polypeptide binding agent or antagonist can readily be determined by assaying its activity. Conservative substitutions are shown in Table 3 below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for functional activity, for example in in vitro cultures in a range of cell types, to determine their ability to bind to extracellular matrix (ECM) proteins, type 1 collagen, type IV collagen and laminin 1.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers, 1993.

Thus, a predicted non-essential amino acid residue in a marker polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the marker polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.

Accordingly, the present invention also contemplates variants of the naturally- occurring polypeptide sequences, of alpha integrin or integrin ligands or their biologically- active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues or functional domains. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a reference polypeptide sequence as, for example, set forth in SEQ E) NO: 2. (amino acid sequence of human ITGA2 polypeptide encoded by SEQ ID NO: 1) Desirably, variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 91, 98, 99% sequence identity to a reference polypeptide sequence as, for example, set forth in SEQ ID NO: 2. Moreover, sequences differing from the native or reference sequence by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which retain the properties of the reference marker polypeptide are contemplated. The subject marker polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to α-subunit encoding polynucleotide sequences, or the non-coding strand thereof. Isoforms or other naturally occurring or sequencing variants of the α-subunit are encompasses.

In some embodiments, variant polypeptides differ from a reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in SEQ ID NO: 2 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution.

A "non-essential" amino acid residue is a residue that can be altered from the wild- type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of a polypeptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. hi other embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a α-subunit polypeptide as, for example, set forth in SEQ ID NO: 2.

The present invention encompasses α-subunits from any mammal or animal (including avian species) subject such as from humans, non-human primates, livestock, laboratory, companion or wild animals. Reference to the ITGA2 polypeptide or α-subunit includes those from any of the above species as well as structural or evolutionary equivalents or homologs thereof. For example, the present invention encompasses an α- subunit portion or its encoding sequence having sequence which has substantially at least about 60% similarity to SEQ ID NO: 2 or at least about 60% identity to SEQ ID NO: 1. Reference to at least about 60% includes 60, 61, 62, 63, 64% and any one of each consecutive numbers in the series to 100%.

Functional derivatives of molecules in genetic form include nucleic acid molecules comprising a nucleotide sequence capable of hybridising to the molecule or its complementary form under low stringency conditions.

The terms "similarity" or identity as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al., Nucl. Acids Res., 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in Molecular Biology John Wiley & Sons Inc, 1994-1998, Chapter 15). The terms "sequence similarity" and "sequence identity" as used herein refer to the extent that sequences are identical or functionally or structurally similar on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, GIy, VaI, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (Marmur et at, J. Mot Biol, 5:109, 1962). However, the T_m of a duplex DNA decreases by I⁰C with every increase of 1% in the number of mismatch base pairs (Bonner et at, Eur. J. Biochem., 46:83, 1974). Foπnamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42⁰C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1 % w/v SDS at a temperature of at least 65°C.

Antisense polynucleotide sequences, for example, are useful in silencing transcripts. Furthermore, polynucleotide vectors containing all or a part of an a subunit gene locus may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with target transcription and/or translation. Furthermore, co-suppression and mechanisms to induce RNAi or siRNA may also be employed. Alternatively, antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells (see, for example, Bourinet et at, The EMBO Journal, 24:315-324, 2005).

A variation on antisense and sense molecules involves the use of morpholinos, which are oligonucleotides composed of morphorine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton et at, Antisense and Nucleic Acid Drug Development, 7:187-195, 1997). Such compounds are injected into embryos and the effect of interference with mRNA is observed.

In one embodiment, the present invention employs agents such as oligonucleotides and similar species for use in modulating the function or effect of a mutation in the ITGA2 gene. The oligonucleotides induce transcriptional or post-transcriptional gene silencing. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the endogenous ligands. The oligonucleotides may be provided directly to a cell or generated within the cell. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding an inhibitor" have been used for convenience to encompass DNA encoding the inhibitor, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

Reference herein to "modulating" includes completely or partially inhibiting or reducing or down regulating all or part of the marker functional activity or enhancing or up regulating all or part marker functional activity or differentiation. Where the molecule is a genetic sequence, its functional activity may be modulated by, for example, modulating its binding capabilities or transcriptional or translational activity, or its half-life. Where the molecule is an encoded polypeptide, its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell or membrane or its enzymatic capability. Modulators are agents which achieve modulation. The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. In one example, the result of such interference with target nucleic acid function is reduced levels of T-type calcium channel a subunit. In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

"Agents" include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate expression thereof. There compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphoro-thioates, phosphoro- dithioates, phosphotri-esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3¹ to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Rational drug design permits the production of structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology, 9: 19-21, 1991). In one approach, one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modelling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modelling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et ah, Science, 249:521-533, 1990). In addition, target molecules may be analysed by an alanine scan (Wells, Methods EnzymoL, 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analysed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. Pharmaceutical compositions for therapy are further contemplated comprising recombinant, synthetic or isolated forms of the herein described agents and one or more pharmaceutically acceptable carriers, diluents or excipients.

The term therapy should be taken as a reference to treatment or prophylaxis of a condition or disease. The term "treating" and "ameliorating" are used interchangeably. The terms "composition" or "agent" or "medicament" refer to a chemical compound that induces a desired pharmacological and/or physiological effect. The term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "compound" is not to be construed narrowly but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof.

The phrases "ameliorating a disease or condition" or "treatment" or "therapeutic" are used in the broadest context and include any measurable or statistically significant improvement in a disease or condition or one or more symptoms or frequency of symptoms of a disease or condition as well as complete recovery from the disease or elimination of a condition, its symptoms or its underlying cause. Conditions may be associated with one or more diseased or they may not be so linked. The amelioration of a condition encompasses any desired physiological or psychological change.

An effective amount of the instant compositions is established best by those skilled in the art. The term "effective amount" of a compound as used herein means a sufficient amount of the agent to provide the desired therapeutic or physiological effect. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount". The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

The polypeptides, nucleic acids, antibodies including humanized antibodies, peptides, chemical analogs, agonists, antagonists or mimetics of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18^th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.

For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. Li preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.

For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilising agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeutically effective amount. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, (supra). Altematively, targeting therapies may be used to deliver the active agent more specifically to certain neural cells of the thalamocortical circuitry, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. The vector could be targeted to neural cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages. The cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent. Alternatively, the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 73 IA and International Patent Publication No. WO 90/07936.

The modulatory agents of the present invention may be chemical agents such as small or large organic or inorganic chemical molecules, peptides, polypeptides including dominant negative forms, modified peptides such as constrained peptides, soluble receptor or extracellular domain molecules or variants thereof, ligands or ligand binding domain mimics foldamers, peptidomimetics, cyclic peptidomimetics, proteins, antibodies or derivatives or deimmunized or humanized forms thereof, lipids, carbohydrates or nucleic acid molecules including antisense or other gene silencing molecules. Small molecules generally have a molecular mass of less than 500 Daltons. Large molecules generally include whole polypeptides or other compounds having a molecular mass greater than 500 Daltons. Agents may comprise naturally occurring molecules, variants (including analogs) thereof as defined herein or non-naturally occurring molecules. Genetic agents such as DNA (gDNA₅ cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs)) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes are useful in some embodiments. Thus, genetic agents include gene-silencing agents such as those above-mentioned. The three dimensional structure of the ce2 subunit has been characterised and accordingly molecular modelling techniques are conveniently used to develop agonists and antagonists. Agonists, for example, have recently been developed using computational modelling, pharmacophore generation and virtual and functional screening as described by Massa et ah, The Journal of Neuroscience, 2<5(20):5288-5300, 2006. In another approach proteolytically stable peptidomimetic agonist ligands of TrkA have been designed (see Maliartchouk et al., Molecular Pharmacology, 57:385-391, 2007). In some embodiments, the antagonist is a peptide, peptidomimetic, foldamer, soluble receptor, extracellular domain, ligand-binding domain or a variant of any of these and wherein said molecule is derived or designed from ITGA2 polypeptide or an ITGA2 -ligand polypeptide. In some embodiments, the antagonist is endorepellin or a functional variant thereof which binds the o2 subunit of the C-2/31 receptor. The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Study Subjects and Preparation

Using a genealogical database at the Menzies Research Institute (MRI) and the records of the Tasmanian Cancer Registry (TCR)₅ families with multiple cases of prostate cancer were identified. All families had at least two affected first-degree relatives, with the largest family comprising 37 affected men, and 10 families having over 11 cases.

Blood samples were available for 131 familial cases in total.

For the purposes of genome-wide linkage analysis, a seven-generation pedigree was identified (Figure 1 - PcTas9), comprising 25 patients diagnosed with histologically confirmed prostate cancer (average age of diagnosis 72, range 50-89). A total of 16 DNA samples were obtained from PcTas9 prostate cancer patients; 9 were extracted from blood samples and 7 from paraffin-embedded tissue. Additionally, 57 DNA samples were available from relatives. DNA was extracted from whole blood or buccal mucosa swabs using the Nucleon Bacc3 (Amersham Biosciences AB, Uppsala, Sweden) and PureGene DNA Isolation Kits (Gentra Systems, MN, USA) respectively. Pathology specimens were obtained from two pathology laboratories located in Southern Tasmania. Using Method E as described by Sato and associates (Sato et al, Diagn. MoI. Pathol., i0(4):265-271, 2001), DNA from a mixture of both normal and tumour cells was extracted from the 7 paraffin embedded tumour blocks suitable for genotyping (Figure 1).

A prostate cancer case control study is also being conducted concurrently by the MRI and is recruiting sporadic prostate cancer cases and unaffected controls. Blood samples, serum samples, physical measures and environmental exposure data are being collected from participating individuals. A total of 412 sporadic prostate cancer cases were identified from the TCR. Eligible cases were men under the age of 75 years diagnosed with histologically confirmed cancer of the prostate during the period 1996 - 2005. Controls were randomly selected from the electoral roll. Eligible controls (319 in total) were age-matched within 5-year age groups to the sporadic cases and self-reported as unaffected with prostate cancer. EXAMPLE 2 Genotyping

Seven prostate cancer patients in family PcTas9 were genotyped with Affynietrix Human Mapping 1OK arrays (indicated with an asterisk in Figure 1), along with 6 first- degree relatives to provide phase information. Genotyping was performed as described by Kennedy and colleagues (Kennedy et al, Nat. BiotechnoL, 21(10): 1233-1237, 2003) using 250ng of genomic DNA. The mean genotype call rate was 95%.

Twenty microsatellites across 5pl3-ql2 (chosen from the Genome Database - http://www.gdb.org/ - see Figure 1) were genotyped in all 73 available DNA samples from

PcTas9 using standard fluorescent PCR techniques and an ABI PRISM 310 Genetic

Analyzer (Applied Biosystems). Microsatellite allele frequencies were estimated from 32

Tasmanian samples comprising spouses of affected cases and unaffected family members.

EXAMPLE 3

Linkage Analysis

PEDCHECK (O'Connell et al, Am. J. Hum. Genet., 63(l):259-266, 1998) was used to identify Mendelian errors in the genotype data. The 1OK SNP data were analysed using the genetic map and Caucasian allele frequencies (Kennedy et al, 2003 (supra); Schaid et al, Am. J. Hum. Genet., 75(6):948-965, 2004). A total of 954 markers in strong linkage disequilibrium (LD) with other markers were removed from the dataset. The family was too large for exact multipoint linkage analysis with the Lander-Green algorithm (Abecasis et al, Nat. Genet., 30(l):97-101, 2002; Gudbjartsson et al, Nat. Genet., 37(10):1015-1016, 2005), and the Markov chain Monte Carlo linkage algorithm Simwalk2 was slow to converge with this number of markers (Sobel et al, Am. J. Hum. Genet., 58(6): 1323-1337, 1996). Linkage analysis was performed using a recently published method that breaks pedigrees into smaller subunits for inference of patterns of identity-by- descent (IBD) sharing between case pairs (Thomson et al, Hum. Genet., 121(3-4):459- 468, 2007). Analysing these smaller pedigrees using Merlin (Abecasis et al, 2002 (supra)) and aggregating results for all case pairs gave an estimate of the NPLp_aj_rs statistic (Whittemore et al., Biometrics, J0(1):118-127, 1994) for the entire pedigree. We have performed simulations, some using the PcTas9 pedigree (Figure 1), to confirm the accuracy of this method for dense marker sets (Thomson et al., 2007 {supra)). Simwalk2 was used to infer haplotypes from microsatellite data and determine the number of cases who inherited the chromosome 5 haplotype. The significance of this haplotype sharing was computed by simulating fully informative markers under the hypothesis of no linkage (gene-dropping) (Sobel et al., 1996 {supra)).

EXAMPLE 4 Prioritising Candidate Genes

Over 90 genes in the region spanned by the chromosome 5 haplotype were prioritised for sequencing using the program 'GeneSniffer' (www.genesniffer.org;

Autogen Limited, Australia). The program builds a list of ranked candidate genes within the specified region using publicly available human and mouse genetic databases, and a supplied list of key words (available on request).

EXAMPLE 5 Sequencing Candidate Genes

The coding regions of eight candidate genes including all exon/intron boundaries and 500bp upstream of the transcription start site were resequenced in PcTas9.4, 9.5, 9.8 and 9.12, who all carried the chromosome 5 haplotype, and in two unrelated controls. The aim was to identify alleles on the chromosome 5 haplotype not carried by the controls. Resequencing was performed using PCR primers designed using the software packages "exon primer" (http://ihg.gsf.de/cgi-bin/snps/seql.pl) and "primer 3" (http://frodo.wi.mit.edu^'). Primer sequences and conditions are available on request. Following PCR amplification, samples were sequenced on an ABI3700 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer's instructions. EXAMPLE 6

Genotyping of Selected Sequence Variants

All familial prostate cancer cases (excluding PcTas9.4, 9.5, 9.8 and 9.12), sporadic cases and controls were genotyped for three sequence variants as outlined below. An allele-specific PCR was used to detect the C>T polymorphism at position -52 of ITGA2 (rs28095 - herein referred to as C-52T). A 172bp fragment was amplified using primers 5'-aatcaggaggggcgggct-3' (SEQ ID NO: 6), 5'-aatcaggaggggcgggcc-3' (SEQ ID NO: 7)and 5'-gcgctgggtttgcagaggtt-3' (SEQ ID NO: 8). The lOμl PCR reaction contained 20ng genomic DNA, Ix GoTaq Green Master Mix (Promega), Ix Q-solution (Qiagen) and 40OnM of each primer. PCR conditions were: 94⁰C for 5 min followed by 30 cycles of 94⁰C for 30 sec, 66°C for 30 sec, and 72⁰C for 30 sec.

A 460bp fragment encompassing the C>T polymorphism at position +807 within exon 7 of ITGA2 (rsl 126643 (CCTCACAAACACATT[C/T]GGAGCAATTCAATAT (SEQ ID NO: 9)) - herein referred to as C807T) was amplified using primers 5'- gatgccttaaagctaccggc-3' (SEQ ID NO: 10) and 5'-taactttcccagctgccttc-3' (SEQ ID NO: 11). The lOμl PCR reaction contained 20ng genomic DNA in Ix GoTaq Green Master Mix (Promega) and 40OnM of each primer. PCR conditions were: 94⁰C for 5 min followed by 30 cycles of 94⁰C for 30 sec, 59⁰C for 30 sec, and 72⁰C for 30 sec. PCR products were digested with 2 units of Hyp 188 1 for 3 hours at 37⁰C and then analysed on 2% agarose gels. Hypl88 1 digestion resulted in fragments of 388bp for the T allele, and 247bp and 141bp for the C allele.

Genotyping the 3'UTR AAC insertion deletion polymorphism (rs3212649 (ATGAATATTGATGTT[-/AAC]AAGAGGGGAAAACAA (SEQ ID NO: 12 and SEQ ID NO: 13) - herein referred to as 3'UTR in/del) involved PCR amplification of the flanking region using a fluorescently labelled forward primer, 5'FAM- gcaactacagaagtggaagtgc-3' (SEQ ID NO: 14), and an unlabelled reverse primer, 5'- tctgtggcaactttggatga-3' (SEQ ID NO: 15). The lOμl PCR reaction contained 20ng genomic DNA in Ix GoTaq Green Master Mix (Promega) and 40OnM of each primer. PCR conditions were as follows: 94⁰C for 5 min followed by 30 cycles of 94⁰C for 30 sec, 59⁰C for 30 sec, and 72⁰C for 30 sec. PCR products were then sized on an ABI310 Genetic Analyser and individuals called as homozygous 134bp (DfD), homozygous 137bp (I/I) or heterozygous (I/D). For all three SNPs, 15% of all samples were re-genotyped to validate genotyping quality.

EXAMPLE 7 Statistical Analysis to detect Association for Selected Sequence Variants

The association analysis excluded all PcTas9 samples with the known chromosome 5 haplotype (cases PcTas9-4, 9-5, 9-8 and 9-12). The M_QLs test (Thornton et al., In press

(supra)) was used to test for differences in allele frequencies at polymorphisms C-52T,

C807T and 3'UTR in/del between familial and sporadic prostate cancer cases and controls.

This test allows for the non-independence of the genotypes of related individuals, regardless of the complexities of the relationships, and also exploits the fact that cases with affected relatives are generally more likely to carry disease susceptibility alleles than cases without affected relatives.

Further exploratory analyses, made no allowance for the non-independence of the familial cases. Logistic regression was used to calculate genotypic odds ratios for the two polymorphisms showing significant association, both adjusted and unadjusted for genotypes at the other polymorphism. Haploview 3.2

(www.broad.mit.edu/mpg/haploview/) was used to examine LD between the polymorphisms, and to perform haplotypic tests of association facilitating the identification of the subject molecular markers.

EXAMPLE 8

Results

The results of the non-parametric analysis of the SNP genome-wide scan are displayed in Figure 2. There was one suggestive linkage peak at 5pl3-ql2 (NPL_paj_rs = 6.72; p = 0.005 using gene-dropping) (Sobel et al, 1996 (supra)). Microsatellite genotyping of the 5pl3-ql2 region in PcTas9 prostate cancer patients and their children provided information on the genotypes of 23 of the 25 cases. Of these 23 cases, 8 were confirmed as sharing a common haplotype, 12 were confirmed as not carrying this same haplotype, and the sharing status of 3 cases could not be determined (Table 5; Figure 1). Given this configuration of genotyped and un-genotyped cases and relatives, the probability of observing a common haplotype shared by 8 or more cases by chance is p=0.0017. The chromosome 5 haplotype extends from marker D5S2506 to marker D5S664, a distance of approximately 14Mb (Figure 1).

Simwalk2 analysis of the entire pedigree using the targeted microsatellite genotyping data gave a maximum non-parametric B Statistic of 2.42 (p=0.0038) over markers D5S1969 and D5S623. A parametric Simwalk2 analysis gave a location score of 1.29 at these same markers.

The 8 patients carrying the chromosome 5 haplotype are clustered into a single branch of the pedigree descendant from individuals 101 and 102. The probability of identifying the same haplotype in 8 or more affected descendants of these individuals is p=0.0003. Simwalk2 analysis of only this branch of the pedigree gave a maximum non- parametric B Statistic of 3.53 (p=0.0003, the same as the p-value from gene-dropping) at marker D5S664 and a parametric location score of 1.95 at markers D5S1969 and D5S623, slightly centromeric to D5S664. The linkage is not of genome-wide significance in this study, however this region was one of five suggested linkage regions identified in a large study conducted by the ICPCG (Xu et ah, 2005 {supra)). Gene-dropping simulations of these five regions showed that the probability of observing segregation in 8 or more cases in one of these regions by chance is p=0.043. This region was examined in more detail by sequencing prioritised candidate genes within the genetic interval. Utilisation of the bioinformatics tool, GeneSniffer, permitted the prioritisation of 13 genes for initial examination. A diagrammatic representation of the 14Mb region of chromosome 5 including the selected genes is shown in Figure 3 (panel A). The coding regions, intron/exon boundaries and promoter sequences of FGFlO,

GHR, DAB2, PPAP2A, PTGER4, EMB, and GZMA were re-sequenced in four PcTas9 cases with the chromosome 5 haplotype (PcTas9.4, 9.8, 9.5, 9.12 in Figure 1), and 2 unrelated controls. No sequence variants, present in all four cases and not in controls, were detected in these genes (data not shown). hi contrast, 15 polymorphisms in the ITGA2 gene were identified as segregating with the shared chromosome 5 haplotype (Table 6). The ITGA2 gene (hgl8 location chr5: 52320913 - 52426366) comprises 30 exons and spans 105288bp (Figure 3, panel B). While 10 of the identified SNPs lie in intronic regions of ITGA2, the remaining five are located in the promoter region (rs28095; C-52T), exon 7 (rsl 126643; C807T), exon 8 (rslO62535 (GATACTTACTGATGC[A/G]AAAGTCCCGTTCCAA (SEQ ID NO: 16)); G873A), exon 27 (rs2303122 (GCGACGAAGTGCTACfA/GJAAAGTAATGGTAGTT (SEQ ID NO: 17)); C3300T), and the 3'UTR of exon 30 (rs3212649; 3'UTR in/del; Table 6). These five SNPs were of immediate interest for genotyping in a second dataset comprising familial cases, sporadic cases and age-matched controls.

The C-52T SNP, located 52 bases upstream of the transcription start site, is positioned between two tandem Spl/Sp3 binding elements and the presence of the T allele has been shown, by in vitro studies, to reduce ITGA2 transcription (Jacquelin et ah, Blood, 97(6): 1721-1726, 2001). However, when this SNP was genotyped in 127 other familial cases, 412 sporadic cases and 319 controls, it showed no association with disease (p=0.49; Table 7). The C807T SNP lies within exon 7, and while it does not alter the amino acid sequence of the protein, there is circumstantial evidence that it is associated with altered function of ITGA2 (Kunicki, Arterioscler. Thromb. Vase. Biol, 22(1): 14-20, 2002). The C807T polymorphism was significantly associated with prostate cancer (p=0.0088; Table 7). There was a more significant difference in allele frequencies between familial cases and controls (p=0.020) than between sporadic cases and controls (p=0.070; Table 7). The G873A SNP in exon 8 is also a synonymous change and as it is in strong LD with C807T (r²=0.97 in the HapMap CEU population of North- West European ancestry (Nature 437: 1299), www.hapmap.org), it was not genotyped.

For the synonymous C3300T polymorphism, no potential functional role has been previously described. Likewise for the 3'UTR in/del in exon 30, the AAC deletion is of unknown function. These two SNPs were perfectly correlated in the PGA European Panel from the SeattleSNPs database (r²=l, pga.gs.washington.edu/). The 3'UTR in/del was genotyped and was significantly associated with prostate cancer (p=0.0009; Table 7). There were differences in allele frequencies both between familial cases and controls (p=0.0018) and between sporadic cases and controls (p=0.015; Table 7). Altogether three SNPs were tested for association in a dataset that did not include the individuals initially identified as carrying the chromosome 5 haplotype, nor individuals who were sequenced across the prioritised genes. After adjusting for multiple testing of three SNPs using the Bonferroni method, the whole-sample p-values quoted above, remain significant. These data suggest that the 3¹UTR deletion allele and the 807T allele both confer risk in a dominant fashion, with similar estimated odds ratios for heterozygous and homozygous carriers of these alleles (Table 8). Odds ratios are higher in familial cases than in sporadic cases for both polymorphisms (Table 8). There is strong LD between the two polymorphisms (1^=0.72; D-0.96) with the 3'UTR deletion allele almost always occurring with the 807T allele, hi the absence of very large sample sizes, this strong LD makes it difficult to separate the individual effects of these alleles, and using a haplotypic test for association, the results were inconclusive (data not shown). However, after adjusting for the 3'UTR in/del and assuming a dominant disease model, the association between the C807T polymorphism and disease is reduced (Table 8). This suggests that the risk from the 807T allele may occur in part because of LD between the two SNPs.

Samples of European ancestry from the HapMap and SeattleSNPs databases were examined to find other candidate SNPs. An examination of these databases revealed 30 and 14 SNPs, respectively, in LD (r²>0.72) with C3300T (i.e. LD greater than that observed between 3'UTR in/del and C807T). A review of these SNPs did not identify any with an obvious functional role and no SNP outside ITGA2 was correlated with an r² >0.35.

EXAMPLE 9 Discussion

Suggestive evidence of linkage at 5pl3-ql2 was obtained following analysis of a genome-wide scan using Affymetrix 1OK SNP arrays in a large Tasmanian prostate cancer pedigree. Subsequent microsatellite genotyping confirmed this finding and identified suggestive linkage, with a shared haplotype carried by at least 8 cases in the pedigree (ρ=0.0017). Nominal evidence for linkage to chromosome 5 has been provided by several previous studies (Camp et ah, 2005 (supra); Smith et ah, 1996 {supra); Wiklund et ah, 2003 (supra); Goddard et ah, 2001 (supra); Hsieh et ah, Am. J. Hum. Genet., 5P(I): 148- 158, 2001) and this region was one of five noted by the ICPCG in a combined analysis of 1,233 prostate cancer families (Xu et ah, 2005 (supra)). Through the use described herein of a gene prioritization tool, re-sequencing, and a follow-up association study, two polymorphisms within the ITGA2 gene, 3'UTR in/del and C807T, were found to be associated with prostate cancer (p=0.0009 and p=0.0088 respectively) in an extended dataset comprising 127 familial cases, 412 sporadic cases and 319 controls.

The ITGA2 gene encodes for the α2 subunit of the α2β 1 integrin receptor, which belongs to a large family of cell surface receptors called integrins. These cell adhesion molecules are responsible for interaction and mediation of signalling events with the extracellular matrix (ECM) proteins type I collagen, type IV collagen and laminin 1 (Koistinen et ah, J. Integrins as Extracellular Matrix Receptors. In: J H, editor. Integrins in Cancer Cell Invasion, Austin, USA: Landes Bioscience; 2006). The α2βl integrin receptor is expressed on many epithelial cells types, including those of the prostate. In normal prostate tissue, expression is restricted to basal epithelial cells (Knox et ah, Differential Am. J. Pathol., 745(1):167-174, 1994). Differentiation of these basal cells into intermediate cells requires them to lose their substratum adhesion, and this is associated with a decrease in expression of integrins, including α2βl (Knudsen et ah, J. Cell. Biochem., PP(2):345-361, 2006).

Studies have shown that α2βl integrin levels vary considerably during tumour development. Prostate cancer stem cells, first described by Collins et al. (2005), were shown to be invasive in Matrigel and are characterized by expression of CD44+/CD133+ and high levels of α2βl integrin (Collins et ah, Cancer Res., 65(23): 10946- 10951, 2005). hi a dataset of primary and metastatic prostate carcinomas, α2β 1 integrin expression was down-regulated in low grade tumours (stage I and II), heterogeneous in intermediate grade tumours and up-regulated in lymph node metastases (Bonkhoff et ah, Hum. Pathol., 2¥(3):243-248, 1993). Up-regulation of α2βl integrin may potentially explain the observation that over 80% of prostate cancer metastases are to bone (Koistinen et al, 2006 {supra); Knudsen et al, 2006 (supra)), since collagen is the main component of bone ECM and the preferred ligand for α2βl integral (Zutter et al, J. Mammary Gland Biol. Neoplasia., 5(2):191-200, 1998). Thus down regulation of α2βl receptor activity or α2 or βl precursor activity or the activity of their encoding genes is used to prevent metastases to bone and/or other collagen rich tissue. Suitable protein antagonists include antibodies, small molecules, aptamers, soluble forms of α2βl receptor α2 or βl precursors, or their ligands and binding regions of these molecules which are known in the art. Methods of identifying and using suitable gene silencing agents are also well developed in the art. There is also considerable evidence that ITGA2 is associated with tumour progression and metastasis in other types of cancer (Koistinen et al., 2006 (supra); Miranti et al, Nat. Cell. Biol., 4(4):E83-90, 2002; Seftor et al, Cancer Res., 5<°(24):5681-5685, 1998; Zutter et al, Proc. Natl. Acad. Sci. U.S.A., 92(16):7411-7415, 1995). α2βl integrin has been demonstrated to mediate cellular invasion in melanoma and squamous cell carcinoma models of tumour metastasis (Zhang et al, J. Cell. Sci., 119(Pt 2):283-291, 2006; Maaser et al, MoI. Biol. Cell., i0(10):3067-3079, 1999). It is further implicated by the finding that the anti-cancer therapeutic agent, endorepellin, mediates its anti- angiogenic activity by binding to α2βl integrin on the surface of the cancer endothelial cells (Bix et al, J. Natl. Cancer Inst., 95(22):1634-1646, 2006). Candidate gene association studies have also linked ITGA2 with cancer risk. Two separate studies of breast cancer and oral cancer have reported an association of the C807T ITGA2 polymorphism with disease risk (Langsenlehner et al, Breast Cancer Res. Treat., P7(l):67-72, 2006; Vairaktaris et al, Eur. J. Surg. Oncol., 32(4):455-457, 2006). In a study comprising 500 sporadic breast cancer cases and 500 controls, the 807C-1648G haplotype was found to decrease risk compared to non-carriers and higher grade breast tumours were significantly associated with the 807T/T genotype (Langsenlehner et al, 2006 (supra)). In addition, the 807T allele was significantly associated with increased risk of oral cancer (p<0.001) (Vairaktaris et al, 2006 (supr-a)). Studies conducted in platelets have demonstrated that the C807T and G873A polymorphisms are associated with changes in receptor levels. The 807T allele is associated with a two-fold higher density of ITGA2 receptor on the platelet cell surface. A predicted enhancer splice element (ESE) at C807T is disrupted in the presence of the T allele (http://pupasuite.bioinfo.cipf.es/). This putative ESE is recognised by splicing factor 2 (sf2) which recruits basal splicing factors and may therefore result in an alternate splice product, hi one embodiment, the 3'UTR in/del polymorphism has a role in regulating ITGA2 IHRNA. The 3'UTR of ITGA2 is unusually large and contains many predicted miRNA target motifs (http://regrna.mbc.nctu.edu.tw/), which suggests it plays an important role in ITGA2 regulation.

Whilst variants within ITGA2 have been significantly associated with breast cancer and oral cancer, to our knowledge this is the first reported association between ITGA2 and prostate cancer. Several genome-wide association studies have now been conducted in large prostate cancer datasets, including the Cancer Genetic Markers of Susceptibility (CGEMS) prostate cancer genome-wide association scan

(www.caintegrator.nci.gov/cgems/browse.do). The study described herein reported no significant associations between prostate cancer and SNPs genotyped within the ITGA2 gene. Although the 3'UTR in/del was not genotyped in their dataset, a marker in strong LD (rs3212601; r²=0.8 in the SeattleSNPs database) was found to have no significant association with disease (p=0.16). Further examination of the CGEMS data revealed a SNP (rs 12515434) within the MOCS2 gene, immediately adjacent to ITGA2, that shows marginal evidence of association with prostate cancer (p=0.047). There is some LD (τ²—035) between rs 12515434 and certain ITGA2 SNPs, however no association was found between rsl2515434 and prostate cancer within our dataset (data not shown).

Although the CGEMS study has not identified ITGA2 as a candidate susceptibility gene, this result is not unusual. Attempts to replicate the significant associations of risk variants within the prostate cancer susceptibility genes ELAC2 (Tavtigian et al, 2001 (supra)), MSRl (Xu et al, Nat. Genet., S2(2):321-325, 2002) and RNASEL (Carpten et al, Nat. Genet., 30(2):181-184, 2002) have also failed to support the original findings (ELAC2: (Camp et al., Am. J. Hum. Genet., 71 (6): 1475-1478, 2002; Rebbeck et al, Am. J. Hum. Genet, <57(4):1014-1019, 2000; Rokman et al, Cancer Res., tfi(16):6038-6041, 2001; Severi et al, J. Natl. Cancer hist, 95(ll):818-824, 2003) MSRl: (Bar-Shira et al, Prostate, <5<5(10):1052-1060, 2006; Noonan-Wheeler et al, Prostate, <5<5(l):49-56, 2006; Sun et al, Prostate, 66(1) :728-737, 2006; Xu et al, Am. J. Hum. Genet., 72(1):2O8-212, 2003) RNASEL: (Chen et al, J. Med. Genet. 40(3):e21, 2003; Maier et al, Br. J. Cancer, P2(6):1159-1164, 2005; Nupponen et al, Genes Chromosomes Cancer, 39(2): 119-125, 2004; Wildund et al, Clin. Cancer Res., 70(21):7150-7156, 2004)). Reasons for this have been widely discussed and include: the heterogeneity of genes contributing to prostate cancer risk in different populations; different selection criteria for each of the study populations examined; and the variable contribution of environmental factors interacting with multiple genetic factors. The Tasmanian population is predominantly of Northern European ancestry (>80% Anglo-Celtic origin; Australian Bureau of Statistics Census, 1996) and as recruitment occurred from one centre in a single state it is a relatively homogeneous dataset both genetically and environmentally.

EXAMPLE 10

Functional role of the ITGA2 at Risk Haplotye andITGAl Gene Regulation

Analysis of the association of disease with the initial 'risk haplotype' (807T, 3¹UTR deletion) formed by the polymorphisms identified in ITGA2 has revealed that the 3¹UTR polymorphism (rs3212649) confers the majority of the measurable risk associated with disease, independently of the C807T. The extensive 3¹UTR of ITGA2 (4.2 kb) surrounding the insertion/deletion SNP was therefore mapped and 6 additional polymorphisms identified and characterised. These additional polymorphisms are rs35440530*, rsl900182, rs6898333, , rs6880055, rs576774800 and rs7725246. The SNP rs35440530* was identified is a CAAA insertion/deletion as shown in Figure 5 in our dataset. This SNP listed in the NCBI database as only a triple base-pair insertion/deletion polymorphism 'AAA', and it is specified that the insertion is at the 3' end of a run of As opposed to the CAAA insertion/deletion identified herein (see Table 9). For clarity, this polymorphism will be referred to as rs35440530 or rs35440530*, however as will be appreciated, reference to this SNP is a reference to the form of the SNP described herein which comprises a CAAA insertion/deletion as shown in Table 9. Together these seven SNPs form the "3'UTR prostate cancer risk haplotype" defined as comprising the following: the AAC deletion at rs3212649, CAAA deletion at rs35440530*, the 'C allele at rsl900182, the 'A' allele at rs6898333, the 'G' allele at rs6880055, the 25bp insertion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'C allele at rs7725246." associated with increased risk of disease. Significant linkage disequilibrium (LD) exists in this region however it is proposed herein that the presence of one or more of these polymorphisms functions to influence ITGA2 gene regulation. Use of modeling software that predicts miRNA binding sequences revealed that two of these SNPs (rs6880055 and rs57674800) are contained within predicted miRNA target motifs (http ://regrna.mbc.nctu.edu.tw/) . The presence of the alternative genoytpe at these sites is predicted to disrupt miRNA binding to the corresponding mRNA sequence and functions to alter gene regulation. The data shown here indicates that the 3'UTR prostate cancer risk haplotype functions to alter gene transcript stability. Reporter gene assays have been performed, essentially as described in Wang G. et al, Am. J. Physiol. Lung Cell MoI Physiol. 284:738- 748, 2003. DNA fragments encompassing approximately 4.2 kb 3'UTR of ITGA2 and representing the risk and non-risk genotypes were generated by PCR using template genomic DNA obtained from selected donors, homozygous for each genotype. These fragments have been sequenced to ensure integrity has been preserved and cloned into the pMIR-REPORT luciferase vector (Ambion). These constructs were transiently transfected into the PC3 human prostate cancer cell line (previously known to express high levels of ITGA2) (Witkoski C et al, J. Cancer Research and Clinical Oncology 119(ll):637-644, 1993). Standard luciferase reporter gene assays were performed to examine influences of genotype on respective mRNA levels. As shown in Figure 6, the construct comprising the 3'UTR prostate cancer risk haplotype demonstrates altered activity versus the wild-type construct (non-risk allele) when transfected into the prostate cancer cell line, PC3 (negative control = no plasmid). The 3'UTR wild-type or non-risk haplotype" is defined as comprising the following: the AAC insertion at rs32112649, the CAAA insertion at rs35440530*, the 'T' allele at rsl900182, the 'G' allele at rs6898333, the 'A' allele at rs6880055, the 25bp deletion 'TATATAAACA ACTTTGTAGG ACTAT' at rs57674800, and the 'A' allele at rs7725246 as shown in Figure 5. Data shown in Figure 6 represents mean luciferase activity for four separate transfections for each construct. These results confirm that the risk haplotype identified regulates gene expression- indeed functions to alter expression of the gene and, presumably modulate o2/31 receptor levels on the cell surface.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Table 1 Summary of sequence identifiers

Table 2 Amino acid sub-classification

Table 3 Exemplary and Preferred Amino Acid Substitutions

Table 4 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid α-ammobutync acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-metliylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylornithine Nmoni

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-metliylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtφ

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionme Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3 -diphenylpropyl) glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(I -nydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3 -indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-niethylproline Dnmpro

N-(I -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylp enicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl) glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diρhenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Table 5

* The possibility of IBD haplotype sharing for these cases cannot be ruled out.

* Not genotyped either due to insufficient DNA or no children participating in the study.

Table 6

Cases Controls

SNP ID Location 9.4 9.8 9.5 9.12 9.122 9.127 rs26679 chr5:52,319,682 CG CG CG CC GG GG rs26680 chr5:52,319,893 TC TC TC TT CC CC rs28095 (C-52T) chr5:52,320,624 TC TC TC TT CC CC rs3212441 chr5:52,373,415 TC TC TT TT CC CC rsl363192 chr5:52,373,590 GT GT GG GG TT TT rsl 126643 (C807T) chr5:52,382,876 TC TC TT TT CC CC rsl 833558 chr5:52,383,068 AG AG AA AA GG GG rs2974987 chr5:52,386,749 AG AG GG GG AA AA rslO62535 (G873A) chr5:52,386,920 AG AG AA AA GG GG rs2303127 chr5:52,391,361 CT CT CC CC TT TT rs2303126 chr5:52,401,669 GA GA GA AA GG GG rs2287871 chr5:52,403,213 CT CT CC CC TT TT O rs984966 chr5:52,404,429 AT AT AA AA TT TT rs2303122 (C3300T) chr5:52,414,784 TC TC TT TT CC CC rs3212649 (3'UTRin/del) chr5:52,422,409 AAC/- AAC/- (-/-) AACx2 AACx2

Table 7

CI — confidence intervals

Table 8

Sporadic Familia Total OR sporadic OR familial OR total OR total*

Genotype Cases I Cases Cases Controls (95% CI) (95% CI) (95% CI) (95% CI)

3¹UTR in/del

I/I 158 38 196 155 1 1 1 1

I/D 198 67 265 127 1.53 (1.12- 2.15 (1.36- 1.65 (1.22- } 1.84 (1.08-3.15)

D/D 56 22 78 37 2.09) 3.42) 2.22)

1.49 (0.93- 2.43 (1.28- 1.67 (1.07-

2.38) 4.58) 2.60)

C807T

C/C 137 34 171 131 1 1 1 1

C/T 204 65 269 138 1.41 (1.02- 1.82 (1.12- 1.49 (1.10- }θ.88 (0.51-1.52) T/T 71 28 99 50 1.95) 2.93) 2.03)

1.36 (0.88- 2.16 (1.19- 1.52 (1.01-

2.10) 3.92) 2.28)

Odds ratios represent increase in odds of disease compared to individuals with I/I or C/C genotypes

CI = confidence intervals

*Odds ratios adjusted for the presence/absence of the disease-susceptibility allele at the other SNP (T or I)

Table 9 Summary of single nucleotide polymorphisms (SNPs) within ITGA2 gene

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Claims

CLAIMS:

1. A method for evaluating prostate cancer in a human subject comprising:

(i) treating a sample from the subject to determine the identity of one or more nucleotides in integrin alpha 2 gene (ITGA2).

2. The method of claim 1 comprising determining the identity of one or more nucleotides in the 3¹UTR of the ITGA2 gene.

3. The method of claim 1 or 2 comprising determining the identity of a subset of selected nucleotides in the 3' UTR of the ITGA2 gene.

4. The method of claim 1 or 3 wherein the nucleotide/s are one or more polymorphisms (SNPs) from the group consisting of: (i) rs3212649;

(ii) rs57674800;

(iii) rs6880055;

(iv) rs35440530*;

(v) rsl900182; (vi) rs7725246;

(vii) rs6898333; and

(viii) rsl126643

5. The method of claim 1 wherein the nucleotide is a T(U), C, A, or G at nucleotide position 902 in SEQ ID NO:1 (rsl 126643), wherein T(U) (or A) indicate the presence of a risk allele and C (or G) indicate the presence of a non-risk allele.

6. The method of claim 4 wherein the selected nucleotide/s is/are one or more of polymorphisms (SNPs) (i) to (iii).

7. The method of claim 6 wherein the selected nucleotide/s is/are (i) and/or (ii).

8. The method of any one of claims 1 to 7 comprising amplifying all or a portion (subsequence) of the ITGA2 gene or probing an amplified portion (subsequence) of the ITGA2.

9. The method of claim 2 comprising amplifying all or a portion (subsequence) of the 3'UTR of ITGA2 or probing an amplified portion (subsequence) of the 3' UTR of ITGA2.

10. The method of any one of claim 1 to 9 wherein the method comprises distinguishing between risk and non-risk alleles comprising sequences selected from the group consisting of:

(a) 3' UTR of ITGA2 at nucleotides 554-556 in SEQ ID NO: 3 having AAC (or

GTT) deleted in a risk allele or AAC (or GTT) present in a non-risk allele (SNP rs3212649);

(b) 3' UTR of ITGA2 at nucleotides 3680-3681 of SEQ ID NO:3 having a 25 bp insertion (TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4) (or its complement) in a risk allele or no 25bp insertion

(TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4) or its complement) in a non-risk allele (SNP rs57674800);

(c) 3' UTR of ITGA2 at nucleotide 3080 in SEQ ID NO: 3 having G (or C) in a risk allele or A (or T) in a non-risk allele (SNP rs6880055); (d) 3' UTR of ITGA2 at nucleotide 3021 in SEQ ID NO: 3 having A (or T) a risk allele or G (or C) in a non-risk allele (SNP rs6898333);

(e) 3' UTR of ITGA2 at nucleotide 2651 in SEQ ID NO: 3 having C (or G) in a risk allele or T (or A) in a non-risk allele (SNP rsl900182);

(f) 3' UTR of ITGA2 at nucleotides 2598-2601 in SEQ ID NO: 3 having CAAA (or TTTG) deleted in a risk allele or present in a non-risk allele in SNP rs35440530*; and

(g) 3' UTR of ITGA2 at nucleotide 4067 in SEQ ID NO: 3 having C (or G) in a risk allele or A (or T) in a non-risk allele (SNP rs7725246).

11. A primer for evaluating prostate cancer in a human subject wherein the primer amplifies a nucleic acid comprising a subsequence of 3' UTR of ITGA2 set out in Figure 5 or Table 9, or a complementary from thereof or a naturally occurring variant thereof.

12. A primer for evaluating prostate cancer in a human subject wherein the primer amplifies a nucleic acid comprising a subsequence of the ITGA2 gene set out in SEQ ID NO:1 or a complementary from thereof or a naturally occurring variant thereof.

13. A nucleic acid probe or primer for use in the evaluation of prostate cancer wherein the nucleic acid is selected from the group consisting of:

(a) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3¹ UTR of ITGA2 having AAC (or GTT) deleted in SNP rs3212649 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having AAC (or GTT) in SNP rs3212649;

3' UTR of ITGA2 having a 25 bp insertion (TATATAAACA ACTTTGTAGGACTAT (SEQ ID NO:4) (or its complement) in SNP rs57674800 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having no 25bp insertion (TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4)) or its complement deleted in SNP rs57674800;

(c) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6880055 but does not hybridise to under high stringency conditions or amplify to a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6880055;

3' UTR of ITGA2 having C (or G) in SNP rs 1900182 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having T (or A) in SNP rsl900182; (e) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6898333 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having G (or C) in SNP rs6898333;

(f) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) deleted in SNP rs35440530* but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of

ITGA2 having CAAA (or TTTG) in SNP rs35440530*; and

(g) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having C (or G) in SNP rs7725246 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs7725246.

14. A nucleic acid probe or primer for use in the evaluation of prostate cancer wherein the nucleic acid is selected from the group consisting of:

(a) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having AAC (or GTT) in SNP rs3212649 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having AAC (or GTT) deleted in SNP rs3212649;

(b) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having a 25 bp deletion (TATATAAACA

ACTTTGTAGGACTAT (SEQ TD NO:4)) or its complement in SNP rs57674800 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having a 25bp insertion (TATATAAACAACTTTGTAGGACTAT (SEQ ID NO:4)) or its complement in SNP rs57674800;

(d) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having T (or A) in SNP rsl900182 but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having C (or G) in SNP rs 1900182;

(e) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the

3¹ UTR of ITGA2 having G (or C) in SNP rs6898333 but does not hybridise to under high stringency conditions or amplify to a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having A (or T) in SNP rs6898333; (f) an oligonucleotide that hybridises under high stringency conditions to or amplifies a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) in SNP rs35440530* but does not hybridise to under high stringency conditions or amplify a nucleic acid molecule comprising a sequence selected from the 3' UTR of ITGA2 having CAAA (or TTTG) deleted in SNP rs35440530*; and

15. The nucleic acid of any one of claims 12 to 14 wherein the nucleic acid is detectably labelled.

16. An array of nucleic acid molecules attached to a solid support, the array comprising a nucleic acid as defined in any one of claims 12 to 14.

17. The array of claim 16 comprising the nucleic acid defined in part a) and/or b) and/or c) of claim 13 or 14.

18. A method of evaluating prostate cancer in a human subject said method comprising treating a sample from a subject to determine the level of expression of IGTA2 mRNA encoding the o2 subunit of c&βl integrin, wherein an elevated level of expression relative to controls is indicative of an increased risk of developing prostate cancer or of developing metastatic prostate cancer.

19. A method of evaluating prostate cancer in a human subject said method comprising treating a sample from a subject to determine the level of expression of IGTA2 mRNA encoding the cQ. subunit of cϋβl integrin, wherein an elevated level of expression relative to controls is indicative that the subject will respond well to prostate cancer treatment or prophylaxis comprising administration of an dλβl integrin antagonist.

20. The method of claim 18 or 19 comprising RT-PCR or Northern analysis.

21. A method of treating or preventing prostate cancer or prostate cancer metastasis in a subject comprising administering an agent which modulates the level or activity of ITGA2 gene or ITGA2 polypeptide (integrin alpha 2, cϋβl integrin) or a ligand or down stream effector.

22. An «2/31 integrin modulator or an odβX integrin-ligand modulator for use in the treatment or prevention of prostate cancer.

23. The modulator of claim 22 wherein the modulator is an antibody that binds to cdβl integrin on prostate cells

24. An c&β\ integrin antagonist or an o2/31 integrin-ligand antagonist for use in the treatment or prevention of prostate cancer or prostate cancer metastasis.

25. An oββl integrin antagonist for use in the prevention of prostate cancer metastasis.

26. The antagonist of claim 24 or 25 wherein the antagonist is endorepellin or a functional variant or analog thereof.

27. The method of claim 21 further comprising evaluating the subject for prostate cancer according to the method of any one of claims 1 to 12 and wherein a subject exhibiting the risk genotype is treated with an α2/31 integrin modulator.

28. The method of claim 21 wherein the agent is an cdβl integrin antagonist.

29. The method of claim 21 wherein the agent is an oCLβl integrin agonist.

30. The method of claim 27 wherein the modulator or antagonist is endorepellin or a functional variant thereof.

31. A kit for evaluating prostate cancer in a subject, said kit comprising at least one reagent that selectively detects the presence or absence of a variation (polymorphism) in the ITGA2 gene.

32. A method of identifying a polymorphism associated with prostate cancer comprising screening the ITGA2 gene or parts thereof from a subject for a polymorphism which segregates with one or more risk alleles in the ITGA2 gene.

33. The method of claim 32 further comprising testing the polymorphic sequence for its ability to modulate ITGA2 gene expression relative to controls.