Patent Publication Number: US-2005142580-A1

Title: Methods and probes for diagnosing a gynaecological condition

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
      This application claims the benefit of the priority of U.S. Provisional Patent Application No. 60/500,072, filed Sep. 3, 2003. 
    
    
     BACKGROUND OF THE INVENTION  
      The present invention relates to DNA sequences useful in the diagnosis of gynaecological conditions such as endometriosis. More specifically, the present invention relates to probes and methods for the utilisation of the differential expression of certain genes in the diagnosis of endometriosis and related conditions.  
      Pelvic endometriosis is a prevalent disease in women of reproductive age. It has a propensity to run a chronic and recurrent course after treatment, leading to debilitating chronic pelvic pain and infertility. The etiology and pathogenesis of endometriosis is controversial. A long held belief postulates that endometrial cells from retrograde menstruation are the origin of the disease. The molecular and cellular events that lead to the implantation and growth of these ectopic endometrial cells and development of endometriosis in these women have yet to be determined. There appear to be some aberrant expressions of growth factors, angiogenic factors and adhesion molecules, and the deficiency of immune system have been postulated to play important roles in development and progression of endometriosis. Endometriosis is most probably a multifactorial/polygenic disorder involving dysregulation of multiple genes in the ectopic endometrial cells.  
      Current methods for diagnosing endometriosis have a number of drawbacks. Diagnosis by physical examination is invasive and may be painful since it is generally performed during early menses, when implants are likely to be largest and most tender. The physician palpates for a fixed, retroverted uterus, adnexal and uterine tenderness, pelvic masses or nodularity along the uterosacral ligaments. A rectovaginal examination is also required to identify uterosacral, cul-de-sac or septal nodules.  
      However, most women with endometriosis have normal pelvic findings, and laparoscopy is necessary for definitive diagnosis.  
      Pelvic ultrasonography, computed tomography and magnetic resonance imaging are occasionally used to identify individual lesions, but these modalities are not helpful in assessing the extent of endometriosis. Even with direct visualization, diagnosis of endometriosis can be difficult. Lesions appear in multiple guises that are at times difficult to interpret. This diagnostic challenge is compounded by the unreliable correlation between clinical manifestations and surgical findings. A patient who is asymptomatic or has very mild symptoms may have extensive disease, whereas an infertile patient may have very few implants.  
      Thus, there exists a need for a more reliable and less invasive method of diagnosing endometriosis. The present invention overcomes or alleviates a problem of the prior art by providing a genetic based method of diagnosis.  
      The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.  
     SUMMARY OF THE INVENTION  
      The applicants have identified a number of DNA sequences that are over expressed or under expressed in cells of a patient having a gynaecological condition such as endometriosis, adenomyosis or endometrioma. Such DNA sequences are identified in the attached Sequence Listing, which forms a part of this application. Accordingly, in one aspect, the present invention provides a DNA molecule or genetic product thereof for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the DNA molecule comprising a nucleotide sequence of one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or equivalents or fragments thereof.  
      In another aspect, the present invention provides primers for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the primers comprising a nucleotide sequence of one or more of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 214, 125, 126, 127, 128, 129, 30, 131, 132, 133, 134, 135, or equivalents or fragments thereof.  
      In a further aspect, the present invention provides a DNA molecule or genetic product thereof for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the DNA molecule comprising a nucleotide sequence of one or more of GenBank accession numbers NM — 001904, AF126110, NM — 000094, NM — 001028, NM — 007104, NM — 021104, X51346, BC006226, NM — 006791, NM — 013293, AK094591, NM — 001533, NM — 005839, NM — 003011, NM — 020306, NM — 004450, BC018111, AF317228, NM — 001798, NM — 005168, NM — 003224, NM — 005000, AY007096, NM — 000196, NM — 025233, X84075, BC004275, NM — 018269, AK001814, XM — 031397, NM — 014179, NM — 152350, NM — 009280, L27560, AK001278, NM — 030939, AK021534, BC014498, BC011980, BC010281, AK026200, AK091133, or equivalents or fragments thereof.  
      Some of these sequences have a high identity with genes with known functions such as Pim-2 oncogenes, IGFBP-5, ribosomal protein L41, propsaposin, fibulin-1, DLX5, 11β hydroxysteroid dehydrogenase type 2, SET, and RhoE.  
      In a further aspect, the present invention provides a method for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition in a subject, the method comprising determining the expression level of a gene comprising a nucleotide sequence described herein in the subject, and comparing the expression level of the gene to the expression level of the same or a similar gene obtained from a reference sample, wherein a positive diagnosis is made if the expression level in the gene is statistically different to that found in the reference sample.  
      In another aspect, the present invention provides a method for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the method comprising the detection of a mutation in a gene, the mutation capable of producing a protein with a higher or lower biological activity than a protein from a non-mutated gene.  
      In yet a further aspect, the present invention provides a probe or primer for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition that is capable of hybridising to a DNA molecule or genetic product thereof as described herein.  
      Also provided is a kit for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition comprising a probe or primer as described herein.  
      Still a further aspect of the present invention provides a method of screening for a compound having efficacy in the treatment or prevention of a gynaecological condition, the method including determining whether the candidate compound is capable of normalizing the expression levels of a gene comprising a DNA molecule as described herein.  
      Another aspect of the present invention provides a method of screening for a compound having efficacy in the treatment or prevention of a gynaecological condition, said method including determining whether the candidate compound is capable of acting as an agonist or antagonist to the protein product of a gene comprising a DNA molecule as described herein.  
      Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  shows a schematic protocol for cDNA subtractive hybridisation. Subtractive hybridization was performed between 1 st -strand cDNA of driver and 2 nd -strand cDNA of tester.  
       FIG. 2  shows subtraction efficiency by depletion of human β-actin and enrichment of the doped gene CAT in the tester cDNA population. The tester cDNA and the eluted cDNA samples were amplified by PCR with β-actin and/or CAT-specific primers before and after subtractions. PCR products were run out on a 1% agarose gel. Cycle 0, 1, 2, and 3 represent the cDNA samples before subtraction and after 1 st , 2 nd , and 3 rd  subtraction, respectively. The high level of β-actin cDNA in the eluted solution (lane 6) demonstrates that the subtractive effect, not degradation, reduced the β-actin cDNA level in the tester.  
       FIG. 3  shows screening procedures and numbers of cDNA clones screened and identified. a, the false positive clones or equally expressed cDNA clones; b, equally expressed or duplicate cDNA clones; c, duplicate clones or cDNA clones containing only human repeat sequence inserts, such as human Alu repeat sequence.  
       FIGS. 4A-4C  show Northern blot and real-time PCR analysis of the cDNA clones selected from the subtractive cDNA libraries, confirming a high agreement between Northern blot hybridization and real-time PCR technique.  FIG. 4A  shows overexpressed candidate genes in endometriosis vs. the paired uterine endometrium confirmed by Northern hybridization. The lanes marked a contain 4 μg of total RNA samples from uterine endometrium. The lanes marked b contain 4 μg of total RNA samples from endometriosis. β-actin was used as control for normalization.  FIG. 4B  shows underexpressed candidate genes in endometriosis vs. the paired uterine endometrium confirmed by Northern hybridization.  FIG. 4C  shows linear regression analysis of gene expression data determined by real-time PCR and by Northern blot analysis using the same paired RNA samples, as shown in  FIG. 4A  and  FIG. 4B .  
       FIGS. 5A-5D  show analysis of differential expression data of 76 candidate genes in 15 paired tissue samples from human endometriosis and autologous uterine endometrium generated by real-time PCR.  FIG. 5A  is a scatter plot of expression data of 76 genes in 15 paired samples after log 2 -transformation. The upper line represents that y=2x, and the lower line represents that y=0.5x.  FIG. 5B  is a TreeView cluster that shows hierarchical clustering of gene expression data of 76 candidate genes (rows) in 15 pairs of clinical samples (columns). Each square represents a ratio of gene expression level in endometriosis to that in uterine endometrium after log 2 -transformation. Color intensity represent overexpression (middle color intensity)-, underexpression (lightest grey) and equal expression (black), respectively, of gene in endometriosis vs. the paired uterine endometrium.  FIG. 5C  is a zoom-in picture for the 14 best candidate genes selected by employing a combination of both the criteria of mean fold-change of 2.0 and P≦0.01 in 15 cases, showing the consistent patterns of their differential expression.  FIG. 5D  shows an analysis of 15 cases by re-clustering based on the expression data of three immediate-early genes EGR1, JUN and JUND. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). The nomenclature for DNA bases as set forth at 37 CFR.s.1.822 are used herein.  
      The applicants have identified a number of DNA sequences that are over expressed or under expressed in cells of a patient having a gynaecological condition such as endometriosis, adenomyosis or endometrioma. Accordingly, in one aspect the present invention provides a DNA molecule or genetic product thereof for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the DNA molecule comprising a nucleotide sequence of one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or equivalents or fragments thereof.  
      It has been shown that the expression of certain genes is altered in a patient suffering endometriosis. The identification of the DNA sequences described herein allows inter alia for the development of diagnostic probes, diagnostic methods, and screening assays to identify compounds useful in treating gynaecological disorders.  
      The skilled person understands that it is not necessary to use the exact sequences defined by SEQ ID NO: 1 to 45. If limited alterations are made to the DNA sequences described herein, the DNA molecules and genetic products thereof are still able to fulfil their functions in the context of the present invention. For example, it is known that many genes are present in alternate forms (i.e. alleles) in different individuals. A probe generated against one allele of a gene is very often able to detect a different allele. Accordingly, the present invention includes within its scope DNA molecules “equivalent” to those described in SEQ ID NO: 1 to 45.  
      Preferably, an equivalent molecule will be at least 90% identical to the sequences described herein. More preferably, an equivalent molecule will be at least 95% identical, and even more preferably it will be at least 97% identical to the sequences described herein.  
      Another indication that two nucleic acid molecules are equivalent is that the two molecules hybridize to each other under stringent conditions when one molecule is used as a hybridization probe, and the other is present in a biological sample. Specific hybridization means that the molecules hybridize substantially only to each other and not to other molecules that may be present in the genomic material. Stringent conditions are sequence dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C. to 2° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. In a preferred form of the invention an equivalent sequence will hybridise to a sequence described herein under stringent conditions.  
      The degeneracy of the genetic code also provides flexibility in the sequences that will be useful in the context of the present invention. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequence that all encode substantially the same protein.  
      The genetic product may be a RNA transcript, or a cDNA derived from the RNA transcript. The genetic product may be a protein. The ability to interchange amino acids in a protein without materially affecting structure or function also provides flexibility in biological systems. For example, substituting a hydrophobic amino acid residue for another hydrophobic amino acid residue will have little effect on the properties of the resultant protein.  
      As a result of the degeneracy of the genetic code and the ability for a protein to tolerate conservative substitutions, a gene coding for a protein in one person can be quite different to that in another. However, there will still be a certain percentage identity between allelic forms of a gene in a given species. It is for this reason that the sequences described herein as well as equivalent sequences are useful in the context of the present invention.  
      It is also understood that fragments of the sequences described herein will be useful in the context of the present invention. Clearly, it is not necessary to utilize the entire sequence to practice the present invention. To be useful as a probe a sequence may be as little as 10 nucleotides long.  
      In a preferred form of the invention the DNA molecule comprises a nucleotide sequence of one or more of SEQ ID NO: 2, 6, 8, 11, 14, 17, 20, 24, 27, 28, 31, 34, 43 or 44 or equivalents or fragments thereof. Applicants have found that genes containing one or more of these sequences displayed a particularly high level of disregulation in subjects suffering from endometriosis.  
      After identifying sequences SEQ ID NOs: 1 to 45 as relevant to the pathogenesis and diagnosis of gynaecological disorders, the applicants performed further sequence analysis to identify the genes from which SEQ ID NO:1 to 45 originate. In screening the GenBank database, applicants have shown that many of SEQ ID NOs. 1 to 45 demonstrate identity with known genes and sequences. The present invention therefore also includes sequences that comprise these genes showing identity to SEQ ID NO 1 to 45. The present invention therefore also provides a DNA molecule or genetic product thereof for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the DNA molecule comprising a nucleotide sequence of one or more of GenBank accession numbers NM — 001904, AF126110, NM — 000094, NM — 001028, NM — 007104, NM — 021104, X51346, BC006226, NM — 006791, NM — 013293, AK094591, NM — 001533, NM — 005839, NM — 003011, NM — 020306, NM — 004450, BC018111, AF317228, NM — 001798, NM — 005168, NM — 003224, NM — 005000, AY007096, NM — 000196, NM — 025233, X84075, BC004275, NM — 018269, AK001814, XM — 031397, NM — 014179, NM — 152350, NM — 009280, L27560, AK001278, NM — 030939, AK021534, BC014498, BC011980, BC010281, AK026200, AK091133, or equivalents or fragments thereof.  
      For example, SEQ ID NO:17 shows identity with the Pim-2 oncogene, which encodes a serine/threonine kinase. It also shows a considerable identity to Pim-1 oncogene, another close-related member of the family. Pim-2 mRNA is highly expressed in various human cancer cells, suggesting the possible involvement of Pim-2 in the transformation processes. Pim-1 and Pim-2 have been identified as common proviral insertion sites in lymphomas induced by MuLV. Pim kinases exhibit strong synergy with c-myc in tumorigenesis, cell proliferation and anti-apoptosis. In human diseases, Pim-1 has recently been shown to be a prognostic biomarker and a co-transcriptional oncogenic factor to myc in prostate cancer. Without wishing to be limited by theory the applicant&#39;s finding of over-expression of Pim-2 in over 90% of endometriosis patients provides the molecular basis for the synergistic transforming effect to take place in the ectopic tissue, conferring upon such tissue survival and progression properties.  
      SEQ ID NO:34 shows identity with a mRNA sequence L27560 (GenBank accession number L27560), which was initially misnamed as IGFBP-5. This gene was closely linked to two members of the IGFBP family and was mapped to chromosome 2q35 between the loci of IGFBP-5 and IGFBP-2 genes. Actually, the 5′-end of the reference mRNA sequence is partially overlapped with the 3′-end (exon 4) of IGFBP-5 gene. Besides structural overlapping, IGFBP-5 also exhibited high over-expression in endometriosis, which follows the same pattern as EA30 gene in the 15 patient cases discussed in the examples below with a correlation coefficient of 0.86 (data not shown). Their expression regulation appears to be governed by a similar mechanism in endometriosis, indicating that EA30 may be functionally related to IGFBP-5 gene.  
      SEQ ID NO:6 has identity with the gene encoding a highly basic ribosomal protein L41, which has been identified as a cellular factor capable of interacting with the CK2β and regulating the CK2 activity. Protein Ser/Thr kinase CK2 is one of the key cellular signals for cell survival, growth and proliferation. CK2 participates in a complex series of cellular functions by modulating the stability and/or activity of various important cellular proteins, such as DNA topoisomerase II, p53, NF-κB, IκB, β-catenin and proapoptosis protein Bid. It has been demonstrated that CK2 exhibits the elevated expression in various cancers and is implicated in tumorigenesis. RPL41 protein has been shown to stimulate the phosphorylation of DNA topoisomerase IIα by CK2 and to enhance the autophosphorylation of CK2α. Without wishing to be limited by theory, applicants propose that over-expression of RPL41 leads to the enhanced activity of CK2 in endometriosis. The up-regulation of RPL41 plays an important role in pathogenesis of endometriosis via modulating the CK2 activity.  
      SEQ ID NO:27 shows identity with Prosaposin, the glycoprotein precursor of saposins A, B, C, and D, which activate lysosomal hydrolysis of sphingolipids. Prosaposin exists in various tissues and body fluids and is especially abundant in the nervous system. Genetic defects in prosaposin have been associated with human lysosomal sphingolipid storage disorders. A recent study on Globoid cell leukodystrophy in a saposin A −/−  mouse model demonstrated that pregnancy dramatically alleviated the clinical and pathological phenotype of the affected mice. The estrogen supplementation produced similar protective effects to pregnancy, indicating that estrogen may, to certain extent, complement the deficiency of saposin A in the mice. Prosaposin was found to be stimulated dose-dependently by estrogen and was secreted by several breast cancer cells. It has been shown that prosaposin mRNA was highly expressed in the adult and embryonic gonads of both male and female mice, suggesting an important role in the reproductive systems. In human breast and ovarian cancer cells, prosaposin interacted with procathepsin D intracellularly and extracellularly, suggesting an involvement in tumor invasiveness and metastasis. In addition, prosaposin treatment of pheochromocytoma cells (PC12) induced extracellular signal-regulated kinases (ERKs) activity, stimulated DNA synthesis, and prevented cell death. Without wishing to be limited by theory, applicants propose that the elevated expression of prosaposin in endometriosis contributes to the survival, proliferation and invasiveness of endometrial cells on the ectopic locations.  
      SEQ ID NO:2 shows identity with the Fibulin-1 protein which is a multifunctional extracellular matrix protein that strongly interacts with fibronectin and suppresses fibronectin-regulated cell adhesion and motility. Over-expression of fibulin-1 in endometriosis suggests the similar mechanism is involved in the pathogenesis of this benign and estrogen-dependent disease.  
      SEQ ID NO:8 shows identity with the DLX5 gene which encodes a homeodomain transcription factor. It is one of six known human DLX homeobox genes, which are required for neurogenesis, skeleton and appendage development, and bone formation. The functions of different members of the DLX family appear to be partially redundant and the targets of DLX regulation include the DLX genes themselves. It has recently been shown that a homeotic transformation of lower jaws to upper jaws occurred in the Dlx5/6−/− mutant mice. Accumulating evidences suggest that DLX5 plays a crucial role in bone development and formation by modulating the gene expression of osteocalcin, COL1A1 and bone sialoprotein. Over-expression of DLX5 in the chicken calvarial osteoblasts stimulated osteoblastic differentiation that does not normally happen in vitro. Ectopic expression of DLX5 in rat osteosarcoma cells also induced up-regulation of fibronectin, suggesting an involvement in cell adhesion. The marked down-regulation of DLX5 expression by up to 92 fold in all 15 endometriosis cases of the following examples indicates that dysregulated DLX5 gene is involved in this pathological process and withdrawal of the effect of DLX5 product facilitates persistence of endometriosis cells in the proliferative state on the ectopic locations by failure of entering end stage differentiation and/or by interference of normal cell adhesion.  
      SEQ ID NO:24 shows identity with the HSD11B2 gene which encodes 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which inactivates glucocorticoid by converting cortisol to cortisone. It plays an important role in modulating glucocorticoid action within a given tissue by pre-receptor regulation, in collaboration with 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). It has been shown that glucocorticoid was able to stimulate expression of P450 aromatase, which converts C19 steroids to estrogens, in human ovarian surface epithelial cell, leiomyoma and adipose tissue. Taken together, these findings indicate that the dysregulated expression of 11β-HSD2 plays a role in the pathogenesis of endometriosis via pre-receptor modulation of glucocorticoid action within the ectopic tissue.  
      SEQ ID NO:14 shows identity with the SET gene which encodes the protein I 2   PP2A , a potent and specific inhibitor of protein phosphatase 2A (PP2A). PP2A is a major protein Ser/Thr phosphatase expressed in all eukaryotic cells. It is involved in diverse cellular processes including cytokine signaling, transcription and translation, and is an important regulator of cell growth and apoptosis. It has been shown that the activity of phosphoprotein phosphatases including PP2A was required for the cAMP-induced expression of steroidogenic acute regulatory (StAR) protein and steroidogenesis. PP2A and SET have also been identified as posttranslational regulators of androgen biosynthesis by Cytochrome P450c17. In addition, PP2A was a binding partner of estrogen receptor α (ERα) and modulated the estrogen-independent activation of ERα by kinase-mediated phosphorylation. More interestingly, it has been demonstrated that Pim kinases were regulated by PP2A at the posttranslational level and inhibition of PP2A activity by okadaic acid stabilized the Pim proteins. Without wishing to be limited by theory, applicants propose that by modulating the PP2A activity, down-regulated SET expression positively contributes to the steroidogenesis and modulates the action of steroid hormones in the endometriotic lesion, whereas it partially alleviates the transforming effect of up-regulation of Pim-2 mRNA (the first candidate gene discussed above) via affecting the protein stability in the pathogenesis of endometriosis.  
      SEQ ID NO:20 shows identity with the RhoE gene encodes RhoE/Rnd3 protein, a member of the Rho family of Ras-related GTPases, which regulate the organization of the actin cytoskeleton in response to extracellular growth factors. RhoE binds GTP, but has no intrinsic GTPase activity and is found constitutively in the activated GTP-bound form. Expression of RhoE in mammalian cells inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions, and induces loss of cell-substrate adhesion leading to cell rounding. RhoE may act to inhibit signaling downstream of RhoA and possess a RhoA antagonistic cell function. It has been shown that RhoA protein is over-expressed in breast cancers as compared to the paired normal tissues and the protein level correlates with tumor progression. Without wishing to be limited by theory, applicants propose that down-regulated RhoE contributes to the pathogenesis of endometriosis by altering cell adhesions and/or by mimicking the effect of enhanced RhoA protein expression in breast cancers.  
      The applicants have defined not only cDNA sequences, but also proteins, and classes of proteins that are involved in the pathogenesis of endometriosis. The present invention therefore includes the use of any sequence encoding a protein that exhibits identity with any one of SEQ ID NO: 1 to 45. Similarities between SEQ ID NO: 1 to 45 with other sequences have been found, and are detailed in Table 1 below.  
               TABLE 1                          Characteristics and chromosome mapping of cDNA clones       identified from subtractive cDNA libraries                                         cDNA   Size   Accession   Map   Sequence       Accession       clone   (bp)   number   locus   homology   Description   number                         Extracellular matrix/cell adhesion proteins                                         EA01   463   BU197985   3p21   CTNNB1   β-catenin (cadherin-associated   NM_001904                           protein)       EA05   396   BU197989   17q21   COL1A1   Collagen, type I, alpha 1   NM_000088       EA29   313   BU198013   21q13   FBLN1   Fibulin-1 isoform D precursor   AF126110       EA52   225   BU198036   3p21.1   COL7A1   Collagen, type VII, alpha 1   NM_000094       EA77   344   BU198061   17q21   COL1A1   Collagen, type I, alpha 1   NM_000088                 Ribosomal proteins                                         EA07   507   BU197991   8q13.2   RPL7   Ribosomal protein L7   NM_000971       EA09   506   BU197993   15q24   RPS17   Ribosomal protein S17   NM_001021       EA10   500   BU197994   4p13   RPL9   Ribosomal protein L9   NM_000661       EA12   587   BU197996   Xq13.1   RPS4X   Ribosomal protein S4, X-linked   NM_001007       EA15   511   BU197999   11q25   RPS25   Ribosomal protein S25   NM_001028       EA16   501   BU198000   6p21.3   RPL10A   Ribosomal protein L10a   NM_007104       EA19   331   BU198003   12q13   RPL41   Ribosomal protein L41   NM_021104       EA61   630   BU198045   19q13.3   RPL13A   Ribosomal protein L13a (a cell   NM_012423                           proliferation inhibitor)                 Transcription regulators                                         EA22   320   BU198006   19p13.1   JUN-D   jun D proto-oncogene   X51346       EA26   527   BU198010   5q31.1   EGR1   Early growth response 1   NM_001964       EA33   333   BU198017   7q21   DLX5   Distal-less homeo box 5   BC006226       EA35   438   BU198019   4p16   CTBP1   C-terminal binding protein 1   XM_042659       EA40   153   BU198024   1p32   JUN   c-jun proto-oncogene   NM_002228       EA63   440   BU198047   15q24   MRG15   MORF-related gene 15   NM_006791                 RNA processing and pre-mRNA splicing factors                                         EA14   369   BU197998   7p15   HTR2A   Similar to  Homo sapiens     NM_013293                           transformer-2 α       EA39   567   BU198023   12q13   FLJ13467   Similar to hnRNP-E2   AK023529       EA53   234   BU198037   19p13.3   FLJ37272   Highly similar to GRG PROTEIN   AK094591       EA54   237   BU198038   19q13.1   HNRPL   Heterogeneous nuclear   NM_001533                           ribonucleoprotein L       EA59   302   BU198043   1p36.11   SRRM1   Serine/arginine repetitive matrix 1   NM_005839                           (a coactivator of pre-mRNA                           splicing)       EA76   169   BU198060   1p35   HPRP8BP   U5 snRNP-specific 40 kDa protein   NM_004814                 Signaling intermediates                                         EA24   531   BU198008   17p13.3   YWHAE   14-3-3 protein, epsilon   NM_006761                           polypeptide       EA27   410   BU198011   9q34   SET   A heat-stable protein phosphatase   NM_003011                           2A-specific inhibitor (I2PP2A)       EA28   292   BU198012   22q13   TOMM22   Translocase of outer mitochondrial   NM_020243                           membrane 22 homolog (yeast)       EA31   175   BU198015   8q22   YWHAZ   14-3-3 protein, zeta polypeptide   NM_003406       EA36   220   BU198020   2p25   Adam17   Rat disintegrin and   NM_020306                           metalloproteinase domain 17       EA43   235   BU198027   2p23   FLJ13786   Highly similar to  Mus musculus     AK023848                           mRNA for ubiquitin conjugating                           enzyme       EA57   223   BU198041   14q24.1   ERH   Enhancer of rudimentary homolog   NM_004450                           ( Drosophila )       EA62   397   BU198046   Xp11.23   PIM2   Pim-2 oncogene   XM_010208                 Cell cycle                                         EA37   319   BU198021   9q22.1-q22.3   SPIN   Spindlin 1   AF317228       EA50   190   BU198034   12q13   CDK2   Cyclin-dependent kinase 2   NM_001798                 GDP/GTP binding proteins                                         EA55   316   BU198039   14q21   ARF6   ADP-ribosylation factor 6   BC008918       EA58   261   BU198042   2q23.3   ARHE   Ras homolog gene family,   NM_005168                           member E       EA70   77   BU198054   20q13.3   ARFRP1   ADP-ribosylation factor related   NM_003224                           protein 1 (Ras-related GTPase)                 Metabolism                                         EA06   401   BU197990   7q31   NDUFA5   NADH dehydrogenase   NM_005000                           (ubiquinone) 1 alpha subcomplex,                           5 (13 kD, B13)       EA21   408   BU198005   19q13.33   LOC126133   Similar to aldehyde   XM_058991                           dehydrogenase 1 family, member                           A2; retinaldehyde dehydrogenase 2       EA60   232   BU198044   16q22   HSD11B2   11-β hydroxysteroid   NM_000196                           dehydrogenase 2       EA64   129   BU198048   17q21   NBP   Similar to nucleotide binding   NM_025233                           protein                 Other cellular functions                                         EA04   178   BU197988   11p11.2   MYBPC   Cardiac myosin binding protein-C   X84075       EA23   159   BU198007   9q22-q31   SEMA4D   Semaphorin 4D   NM_006378       EA34   361   BU198018   1q23   ATP1B1   ATPase, Na+/K+ transporting,   NM_001677                           beta 1 polypeptide       EA41   127   BU198025   10q22.1   PSAP   Prosaposin   XM_045140       EA44   264   BU198028   2p25.3   SIPL   A hepatic factor supporting   NM_018269                           hepatitis C virus replication       EA78   301   BU198062   3p25   IMAGE:   Similar to ARPC4   BC012596                       429689                 Unknown function                                         EA02   605   BU197986   10q21   IMAGE:   Uncharacterized   BC018658                       4082362       EA03   330   BU197987   10p15   IMAGE:   Uncharacterized   BC015987                       4096273       EA11   391   BU197995   2p22   FLJ10952   Uncharacterized   AK001814       EA13   379   BU197997   1p36.32   KIAA0495   Uncharacterized   XM_031397       EA17   196   BU198001   1p36.12   HSPC157   cDNA clone from CD34+ stem   NM_014179                           cells       EA18   473   BU198002   17p11.2   TSAP19   mRNA activated in tumor   AJ012499                           suppression       EA25   493   BU198009   18q11.2   Ss18   Similar to  Mus musculus  synovial   NM_009280                           sarcoma translocation,                           Chromosome 18       EA30   207   BU198014   2q35   LOC151361   Uncharacterized   XM_098048       EA32   290   BU198016   20q13.33   FLJ20602   Hypothetical protein   AK000609       EA38   195   BU198022   1q23.3   FLJ10416   Uncharacterized   AK001278       EA42   373   BU198026   6p22   FLJ12619   Hypothetical protein   NM_030939       EA45   229   BU198029   3p14   A430107N12   Uncharacterized, from RIKEN   AK020781                             Mus  full-length enriched library       EA46   223   BU198030   7p22.2   IMAGE:   Uncharacterized   BC027714                       4128750       EA49   294   BU198033   2q33.1   FLJ13448   Hypothetical protein   BC022453       EA51   246   BU198035   12q14   FLJ11472   Uncharacterized   AK021534       EA56   507   BU198040   11q12   IMAGE:   Uncharacterized   BC014498                       4856273       EA65   135   BU198049   5q22   IMAGE:   Uncharacterized   BC013250                       3867502       EA66   254   BU198050   1p34.1   SP192   Hypothetical protein SP192   NM_021639       EA67   420   BU198051   5q23   FLJ21409   Uncharacterized   AK025062       EA68   293   BU198052   5q32   HLCDGP1   Down-regulated in lung cancer   NM_018548       EA69   217   BU198053   12p13   IMAGE:   Uncharacterized   BC011980                       3860421       EA71   204   BU198055   16p12   IMAGE:   Uncharacterized   BC010281                       3048642       EA72   374   BU198056   2q34   KIAA0981   KIAA0981 protein   XM_028867       EA74   257   BU198058   4q21   FLJ22547   Uncharacterized   AK026200       EA75   322   BU198059   22q12   FLJ33814   Uncharacterized   AK091133                 Novel genes (match human genomic sequence)                                         EA08   491   BU197992   12q15   BAC clone   Novel   AC121761       EA20   367   BU198004   2p21   BAC clone   Novel   AC073082       EA47   222   BU198031   8q23   BAC clone   Novel   AP000427       EA48   288   BU198032   5p13   BAC clone   Novel   AC008768       EA73   342   BU198057   9q34   BAC clone   Novel   AL354944                  
 
      In another aspect the present invention provides a method for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition in a subject, the method comprising determining the expression level of a gene comprising a nucleotide sequence of any of SEQ ID NOs: 1 to 45, or equivalents or fragments thereof in the subject, and comparing the expression level of the gene to the expression level of the same or a similar gene obtained from a reference sample, wherein a positive diagnosis is made if the expression level in the gene is statistically different to that found in the reference sample.  
      The method may be used to diagnose a new gynaecological condition in a subject, or provide further information on an existing gynaecological condition. In both cases the method can be used to provide prognostic information for use by the clinician to assist in the management of the condition.  
      The method could use any one or more of a number of biological samples from the subject as a source for determining the expression level of the gene. In a preferred embodiment of the method the biological sample is tissue biopsy material from the vagina, uterus, cervix, fallopian tube or ovary. The biological sample may also be a fluid such as blood, saliva, urine, or a secretion of the reproductive tract. Preferably the sample is a biopsy of uterine endometrial tissue.  
      Where determination of the expression level involves isolating or purifying a polynucleotide or protein from the biological sample, the skilled artisan will be able to select an appropriate method. As an example, suitable techniques are described by Maniatis, T., Fritsch, E., F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. N.Y. (1989)  
      The expression level of the gene may be determined by any method familiar to the skilled person. One way of determining the expression level of a gene is to quantitate the amount of mRNA that has been transcribed from the relevant gene. This can be achieved by Northern blotting techniques for example. The quantitation could be determined on an absolute scale, or a relative scale.  
      The method of diagnosis requires that a judgement as to whether the gene is under expressed, or over expressed by comparison to a reference value for the same or similar gene. The reference value could be obtained from another sample from the subject that would be unlikely to be involved in a gynaecological condition. In a preferred form of the invention the reference value is obtained from eutopic endometrium taken from the subject.  
      Alternatively, the reference value could be an expressing level or average of a number of expression levels determined using biological samples taken from one or more subjects not suffering a gynaecological condition.  
      In a preferred embodiment of the method a positive diagnosis is made if at least a two-fold difference is noted between the expression level of the test sample and the reference sample. Based on further experience with the diagnostic method the skilled person may arrive at other cut off values. However, determining more appropriate cut off values is well within the ability of the skilled person.  
      In a preferred embodiment of the invention the method considers the expression level of a gene that is over expressed by at least a factor of two (i.e. SEQ ID NO: 2, 6, 11, 17, 27, 28, 34, 43, or 44). In a further preferred embodiment, the method considers the expression of a gene that is under expressed by a factor of at least two (i.e. SEQ ID NO: 8, 24, 14, or 20). In a further preferred embodiment, the method considers the expression of a gene that is under expressed by a factor of 15 (i.e., SEQ ID NO: 8).  
      In a preferred form of the invention the expression level is determined by analysis of a gene transcript, such as Northern blotting, quantitative PCR or sequencing.  
      In a further preferred form of the invention the expression level is determined by analysis of a protein encoded by the gene. In a more highly preferred embodiment the analysis of the protein is by Western blot, ELISA, surface plasmon resonance, or amino acid sequencing.  
      The skilled person will understand that the expression of more than one gene is likely to provide a more definitive picture of the gynaecological condition. Accordingly, in a preferred method the expression levels of at least 5 genes are considered. In a more highly preferred method the expression levels of at least 10 genes are considered.  
      Another aspect of the present invention provides a method for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition, the method comprising the detection of a mutation in a gene, the mutation capable of producing a protein with a higher or lower biological activity than a protein from a non-mutated gene.  
      It is possible that while a certain gene involved in a gynaecological condition is not under expressed or over expressed, the protein product has an amino acid sequence that affords it a higher or lower than normal biological activity. Thus, the expression level may appear normal, but a gynaecological condition will still result because of the abnormally high or low biological activity of the protein.  
      In another aspect the present invention provides a probe or primer for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition that is capable of hybridising to a DNA molecule or genetic product thereof as described herein.  
      Nucleic acid probes and primers may readily be prepared based on the nucleic acids provided by this invention. A probe may comprise an isolated polynucleotide attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, e.g., in Sambrook et al. (1989).  
      Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.  
      Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (1989). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides of the gene of interest will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.  
      Given the DNA sequences provided in the instant specification, the skilled person is adequately enabled to produce a probe or primer useful in the methods described herein.  
      In a preferred form of the invention the primers comprise nucleotide sequences of one or more of SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 214, 125, 126, 127, 128, 129, 30, 131, 132, 133, 134, 135, or equivalents or fragments thereof.  
      Furthermore, a skilled person could deduce the protein that would be translated from the transcript, synthesise the protein (or fragment of the protein), and use it to generate antibody probes specific for that protein.  
      The present invention contemplates the use of one or a plurality of sequences. In a preferred embodiment of the invention a plurality of sequences are screened for abnormal expression in the patient. A clinician would be more convinced that a patient was in fact suffering from a gynaecological condition if abnormal expression levels were seen for a number of candidate genes.  
      In another aspect the present invention provides a kit for monitoring the progression of, or diagnosing, or determining the severity of a gynaecological condition comprising a probe or primer as described herein.  
      In another aspect the present invention provides a method of screening for a compound having efficacy in the treatment or prevention of a gynaecological condition, the method including determining whether the candidate compound is capable of normalizing the expression levels of a gene comprising a DNA molecule as described herein.  
      The present invention is not limited to the diagnosis or prognosis of gynaecological conditions, but will have use in the treatment of such conditions. The sequences identified by the applicants as important in pathogenesis point to a number of genes that are involved in the disease process. This allows further investigations into the identification of compounds that are able to modulate expression of the relevant genes allowing the expression levels to return to normal. For example, if a gene is over expressed in endometriosis, the aim would be to decrease expression. This could be accomplished by gene therapy methods (such as antisense technology). If a gene is under expressed, gene therapy could be used to augment production of the gene product.  
      Similarly, it would be possible to identify compounds that are capable of modulating the biological activity of a protein gene product encoded by a gene described herein. Another aspect of the present invention therefore provides a method of screening for a compound having efficacy in the treatment or prevention of a gynaecological condition, said method including determining whether the candidate compound is capable of acting as an agonist or antagonist to the protein product of a gene comprising a DNA molecule as described herein.  
      The invention will be applicable to many diseases and conditions of gynaecological origin that relate to the ectopic or uncontrolled growth of uterine tissue. Preferably, the uterine tissue is endometrium.  
      The gynaecological condition may be related to a disorder of any pelvic tissue such as uterine, fallopian, ovarian, cervical or vaginal tissue. It will be understood that tissues and other than the reproductive tissues may also be involved. For example, the ectopic growth of uterine tissue may occur in other organs or structures of the pelvis such as the ureters, bladder, urethra, kidney, peritoneum, mesentery, abdominal wall, diaphragm, stomach, liver, pancreas, intestine, rectum, blood vessel, or connective tissue. Conditions affecting these organs or structures are therefore included in the scope of the present invention.  
      In a preferred form of the invention the gynaecological condition to which the present invention applies includes, but is not limited to, endometriosis, adenomyosis and endometrioma.  
      The invention will now be further described in the following non-limiting examples.  
     EXAMPLES  
     Example 1  
     Materials and Methods  
      Tissue specimens. Tissue biopsies of pelvic endometriosis and normal uterine endometrium were collected in pair from 15 patients undergoing laparoscopy or hysterectomy for endometriosis (Table 2). Portions of the tissue specimens were stored in liquid nitrogen immediately for this study while the remaining were used for histo-pathological diagnosis. The patients either had never received any hormonal treatment or had ceased any hormonal medication for at least 6 months before surgery. The patients were categorized by menstrual phases and severity of endometriosis. The proliferative (days 4-14) and secretory (days 16-28) phases of menstruation was determined by the patients&#39; menstrual history and histo-pathological assessment of the endometrium. The severity or stage of evolution of endometriosis was based on surgical pathological findings according to the revised American Fertility Society classification system (Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril. 1997 May; 67(5):817-21). For analysis of gene expression profiles, the 15 pairs of samples were classified into two groups: (A) early disease (clinical stage I &amp; II) and (B) advanced disease (clinical stage IlI &amp; IV).  
               TABLE 2                          Characteristics of clinical tissue biopsies used for gene       expression profiling by real-time PCR                                     Patient                       No.           Stage of       Patient group   &amp; symbol   Age (yr)   Cycle phase   endometriosis               Early disease    1 (I-P1)   33   Proliferative   I            2 (I-P2)   32   Proliferative   I            3 (II-P1)   28   Proliferative   II            4 (II-P2)   32   Proliferative   II            5 (II-P3)   34   Proliferative   II            6 (II-S1)   32   Secretory   II       Advanced disease    7 (III-P1)   50   Proliferative   III            8 (III-P2)   43   Proliferative   III            9 (III-P3)   35   Proliferative   III           10 (III-N)   29   Not available   III           11 (IV-P1)   46   Proliferative   IV           12 (IV-P2)   41   Proliferative   IV           13 (IV-S1)   34   Secretory   IV           14 (IV-S2)   39   Secretory   IV           15 (IV-S3)   36   Secretory   IV                  
 
      RNA preparation. The fresh-frozen tissue was weighted, rinsed with ice-cold PBS solution and minced on ice before RNA purification. Total RNA was prepared from 7-50 mg fresh-frozen tissue specimens using Triazole™ Reagent according to the protocol recommended by the manufacturer (Gibco BRL).  
      1 st -strand and 2 nd -strand cDNA synthesis. mRNA was purified from 30-40 μg of total RNA using the Dynabeads® Oligo (dT) 25  (DYNAL) reagent and 1 st -strand cDNA was synthesized (42° C., 50 min; 50° C., 30 min) with SuperScript II™ Reverse Transcriptase (Gibco BRL, 400 units) reagent using Dynabeads® Oligo (dT) 25  reagent as RT primer according to the protocol recommended by the manufacturer (DYNAL). All the reactions involving Dynabeads® reagent were performed on a roller mixer. After cDNA synthesis, the unprimed Oligo (dT) 25  on the Dynabeads® reagent were removed by T4 DNA polymerase (New England Biolabs, 3 units, room temperature, 1 hour) (9). 1 st -strand cDNA on Dynabeads® reagent was tailed with poly(dA) n  by Terminal Deoxynucleotidyl Transferase (Gibco BRL, 15 units, room temperature, 1 hour). A 2 nd -strand cDNA was then synthesized with Taq DNA polymerase (Qiagen) using a one-base anchor oligonucleotide HT 26 V (AAGCTTTTTTTTTTTTTTTTTTTTTTTTTTV, V: A, G or C; SEQ ID NO: 136) as primer (94° C., 3 min; 55° C., 2 min and 68° C., 15 min). The 2 nd -strand cDNA was then eluted in 30 μl TE buffer with 0.6 μg carrier tRNA (Gibco BRL) from the 1 st -strand cDNA Dynabeads at 95° C., 2 min. The 1 st -strand cDNA Dynabeads® reagent was then removed with a magnet (Dynal MPC-E-1).  
      Subtractive cDNA library construction. Subtractive hybridization was performed in a small volume (≦30 μl) based on the solid-phase cDNA using a similar protocol as described by Lönneborg et al. 1995 PCR Methods Appl., 4(4):S168-176, with several modifications.  FIG. 1  shows the schematic protocol for cDNA preparation and subtractive library construction used in this study. Briefly, the main modifications include: (a) 2 nd -strand cDNA was used as tester instead of mRNA; (b) subtractive progress was monitored by PCR amplification of β-actin gene in tester solution after each cycle of subtraction. Efficacy of the protocol was evaluated by duplex PCR of β-actin and CAT genes, representing common and differential sequences, respectively, in the differential cDNA model system, which consisted of a driver—1 st -strand cDNA Dynabeads® reagent and a tester—2 nd -strand cDNA from the same RNA doped with an extra CAT cDNA. PCR primers used for β-actin and CAT genes were as follows: (a) β-actin forward primer, 5′-ATGGATGATGATATCGCCGC-3′ SEQ ID NO: 137; (b) β-actin reverse primer, 5′-CTAGAAGCATTTGCGGTGGA-3′ SEQ ID NO: 138; (c) CAT forward primer, 5′-GACATGGAAGCCATCACAGAC-3′ SEQ ID NO: 139; and (d) CAT reverse primer, 5′-CGACCGTTCAGCTGGATATTAC-3′ SEQ ID NO: 140.  
      After subtractive hybridization using cDNA samples prepared from endometriosis and paired uterine endometrium, the remaining 2 nd -strand tester cDNA with a poly(dA) n -tail in the hybridization solution was isolated by using Dynabeads® Oligo (dT) 25  reagent. The subtracted cDNA molecules were globally amplified by PCR using HT 26 V universal primer. The PCR product was purified with the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech). The purified PCR product was then cloned into a pCR-XL-TOPO TA vector (Invitrogen), and transformed into TOP10 competent cells to generate a subtractive cDNA library. [0081] Screening of subtractive cDNA libraries by differential hybridization. 738 colonies were random-selected from the subtractive libraries for differential screening with colony and/or dot blot hybridization. About 400 colonies were prescreened by colony hybridization prior to dot blot hybridization. The rest colonies were directly screened by PCR and dot blot hybridization. For dot blot hybridization, cDNA inserts were amplified by colony-PCR using the vector-specific primers (T7 primer: 5′-TAATACGACTCACTATAGGG-3′ SEQ ID NO: 141; M13 Reverse primer: 5′-GGAAACAGCTATGACCATG-3′ SEQ ID NO: 142). PCR product was mixed with equal volume of 0.6 N NaOH to denature dsDNA. 2 μl of denatured DNA sample was transferred to Hybond-N+™ nylon membrane (Amersham Pharmacia Biotech) with a micropipettor. A 1128-bp human β-actin cDNA (coding region) and a vector without insert were used as the positive and negative controls, respectively. The blotted membranes were then neutralized with 0.5 M Tris-HCl (pH 7.5) and cross-linked using an UV Stratalinkert instrument (Stratagene). The cDNA probes prepared from paired endometriosis and endometrium samples were labeled with Digoxigenin-11-dUTP (Boehringer Mannheim). Colony and dot blot hybridization with the DIG-labeled probes and their chemiluminescent detection were performed according to the protocol recommended by the manufacturer (Boehringer Mannheim).  
      DNA sequencing. DNA sequencing was performed with ABI Prism BigDye™ Terminator Cycle Sequencing Ready Reaction Kit and ABI Prism® 310 Genetic Analyzer (PE Applied Biosystems). Cycle sequencing reaction was carried out with GeneAmp® PCR System 9700 (PE Applied Biosystems) using T7 and/or M13 Reverse sequencing primers.  
      Northern analysis. Total RNA samples (4-8 μg) were electrophoresed on 1.2% formaldehyde denaturing agarose gel prior to overnight capillary transfer to BM nylon membranes (positively charged, Boehringer Mannheim). The previously isolated cDNA clones were purified with Wizard® plus SV Minipreps DNA Purification System (Promega). DIG-DNA probes were then prepared from the purified cDNA clones by PCR using the vector-specific primers. The RNA blots were hybridized with DIG-DNA probes in DIG Easy Hyb solution (Boehringer Mannheim) at 50° C. overnight, followed by washes of 2×5 min in 2×SSC, 0.1% SDS at room temperature and 2×15 min in 0.1×SSC, 0.1% SDS at 68° C. under constant agitation. Chemiluminescent detection of the hybridized blots was performed according to the protocol recommended by the manufacturer (Boehringer Mannheim). Northern blots were quantified with densitometry and the expression data for target genes were then normalized to β-actin mRNA levels for further analysis. The extent of differential expression of these genes was determined by the fold-change of expression levels in endometriosis versus the paired uterine endometrium after normalization.  
      Gene expression profiling by real-time PCR. 4 μg of total RNA from each samples were treated with RNase-free DNase I (Gibco BRL) to eliminate the contaminant genomic DNA. The mixtures were directly used for reverse transcription with random hexamer primers and SuperScript First-Strand Synthesis System (Gibco BRL). Real-time PCR was performed with SYBR® Green Master Mix on an ABI Prism® 7700 Sequence Detection System according to the manufacturer&#39;s protocol (PE Applied Biosystems). The cDNAs were amplified by incubation for 10 minutes at 95° C. to activate the Hot Start AmpliTaq Gold® DNA polymerase reagent, followed by 45 cycles of denaturation at 94° C. for 30 seconds, annealing at 56° C. for 1 minutes and extension at 68° C. for 1 minutes. 15 ng of cDNA (total RNA equivalent) was used for each PCR reaction performed in 20 Eli with Optical 96-Well Reaction Plates (PE Applied Biosystems). Melting curves were generated after amplification to check the PCR specificity. Amplicon size and reaction specificity were further confirmed by electrophoresis on a 1.5% agarose gel and a single PCR product with expected size should be observed. The changes in fluorescence of the SYBRO® Green I dye in each cycle were monitored by ABI Prism® 7700 system, and the threshold cycle (C T ), which is defined as the cycle number at which the amount of amplified target reaches a fixed threshold, was obtained for each gene in each sample. Both primer sets for β-actin and 18S rRNA were included in each plate of PCR reactions as endogenous control and β-actin was used to normalize the quantity of RNA used. The C T  value of β-actin was subtracted from that of each target gene to obtain a ΔC T  value. The relative mRNA level of each target gene in each pair of samples was determined by 2 −ΔΔCT , where 
 
ΔΔ C   T   =ΔC   T [Gene( x ) endometriosis   ]−ΔC   T [Gene( x ) endometrium ],
 
 ΔC   T [Gene( x ) endometriosis]   =C   T [Gene( x ) endomethosis   ]−C   T [(β-actin) endometriosis ],
 
and Δ C   T [Gene( x ) endometrium   ]=C   T [Gene( x ) endometrium   ]−C   T [(β-actin) endometrium ].
 
      The C T  values of each gene in endometriosis and the paired endometrium samples were determined in triplicate experiments and the mean C T  values were used for analysis. The real-time PCR reactions were repeated if the coefficient of variation (CV) for any C T  values was more than 4%.  
      Primers used for real-time PCR. Primers for target genes were designed based on the sequences of the cDNA clones and synthesized by OPERON (USA). The nucleotide sequences for 78 selected cDNA clones (EA01-EA78) and 76 pairs of primers (corresponding to clone EA01-EA76) have been submitted to the GenBank (GenBank accession number: BU197985-BU198062) and will be available at the Web site: http://www.ncbi.nlm.nih.gov. The information provided therein is incorporated herein by reference. The primer sequences for β-actin mRNA and 18S rRNA are as follows: (a) β-actin forward primer: 5′-CCAGCACAATGAAGATCAAGATCA-3′ SEQ ID NO: 143; (b) β-actin reverse primer: 5′-GGGCCGGACTCGTCATACT-3′ SEQ ID NO: 144; (c) 18S rRNA forward primer: 5′-TCGCTACTACCGATTGGATGGT-3′ SEQ ID NO: 145; and (d) 18S rRNA reverse primer: 5′-CACCTACGGAAACCTTGTTACGA-3′ SEQ ID NO: 146.  
      Data analysis. The differential gene expression measured by the relative quantitative values (fold changes) given by 2 −ΔΔCT  for each target gene in each pair of samples were analyzed and displayed by hierarchical clustering algorithm after logarithmic (log 2 ) transformation using the Cluster and TreeView software (Eisen et al, 1998 Proc. Natl. Acad. Sci. USA, 95:147863-14868). The genes and clinical samples were grouped on the basis of similarities of gene expression patterns. Statistical analysis for difference of gene expression data between normal endometrium and endometriotic tissue samples was performed with paired Student&#39;s t test. Analyses with a P value of 0.05 or less were considered to be statistically significant.  
      Chromosome mapping. The candidate genes of interest were mapped to chromosomes by searching human genome, LocusLink and UniGene databases at the Web site: http://www.ncbi.nlm.nih.gov.  
     Example 2  
     Subtractive Hybridization, Library Construction and Screening with RNA Samples from Paired Endometrium and Endometriosis Tissues  
      The efficacy of the subtractive protocol was confirmed experimentally ( FIG. 2 ). The subtractive hybridization in two directions to enrich the genes over-expressed or underexpressed in endometriosis tissue generated a large number of cDNA clones. 738 colonies were screened by colony PCR and/or differential hybridization to remove those false positive clones, and duplicate or equally expressed genes. 108 cDNA clones were identified for further study with DNA sequencing ( FIG. 3 ).  
     Example 3  
     DNA Sequencing Analysis, Gene Identities and Chromosome Mapping  
      DNA sequencing analysis through the GenBank databases for 108 cDNA clones isolated from the libraries revealed that 78 contained different cDNA inserts while the other 30 were duplicate clones or had the same inserts with different orientation and were therefore excluded. Of 78 cDNA clones, 48 were identical with or very similar to known genes in the GenBank databases, and 25 were matched to unknown genes, including uncharacterized human or mouse mRNA and hypothetical proteins. The remaining 5 clones matched human genomic sequences with no similarity with any known cDNA or mRNA, i.e. novel genes. The 48 known genes included 5 genes for extracellular matrix/cell adhesion proteins, 8 for ribosomal proteins, 6 for transcription regulators, 6 for RNA processing and pre-RNA splicing factors, 8 for signaling intermediates, 2 for cell cycle, 3 for GDP/GTP binding proteins, 4 for metabolism and 6 for other cellular functions. All these 78 genes were mapped to chromosomes by searching human genome, LocusLink and UniGene databases (see Table 1 above).  
     Example 4  
     Northern Analysis  
      To verify the differential expression of the genes, those cDNA clones generating strong signals during differential screening were analyzed with Northern blot hybridization on the same pair of original RNA samples used for subtractive hybridization. This confirmed the differential expression of 18 genes in endometriosis versus the paired uterine endometrium ( FIGS. 4A-4C ). Of these 18 genes, 8 were over-expressed ( FIG. 4A ) and 10 were under-expressed ( FIG. 4B ) in the endometriotic sample. Notably, there was a marked over-expression in EA26 (EGR1), EA40 (JUN) and EA62 (PIM2) genes and under-expression in EA18 (TSAP19), EA27 (SET), EA35 (CTBP1) and EA61 (RPL13A) genes. Northern blots were quantified with densitometry and the expression data for target genes were then normalized to β-actin mRNA levels ( FIG. 4A ) for further analysis.  
     Example 5  
     Gene Expression Profiling by Real-time PCR  
      The CYBR Green-based real-time PCR technique was used for gene expression profiling in multiple clinical samples from patients (Table 2) with endometriosis at different stages (I-IV) after an evaluation study confirming a high agreement between real-time PCR and Northern blot hybridization (r 2 =0.986) ( FIG. 3  at (c)). Some 8,000 real-time PCR reactions were performed to analyze the expression profiles of 76 out of 78 candidate genes in 15 pairs of clinical tissue samples.  
      To obtain a general idea of differential regulation of 76 genes in 15 paired samples gene expression data generated by real-time PCR were displayed on a scatter plot ( FIG. 5A ). Of 1140 data points, 649 lay between the red and green lines and therefore their expression ratios in endometriosis samples vs. the paired uterine endometrium are within the range from 0.5 to 2.0. The other 491, representing 43% of the total data points, lying outside this range indicate that the genes were differentially expressed by over 2 fold in these paired clinical samples.  
      Two methods were used to identify differentially regulated genes:  
      (1) Using an arbitrary cutoff criteria. Of the 76 candidate genes, 30 (39%) showed consistent differential expression in more than 70% of cases (at least 11 out of 15). In fact, consistent differential expression was seen in 15 (100%) cases for 2 genes, in 14 (93%) cases for 3 genes, in 13 (87%) cases for 8 genes, in 12 (80%) cases for 9 genes, and in 11 (73%) cases for another 8 genes. Raising the cutoff threshold to 2-fold difference between endometriosis and autologous endometrium tissues, 14 (18%) of 76 genes were consistently differentially expressed in at least 9 (60%) of 15 cases.  
      (2) Using significant statistical difference as criteria. A quantitative assessment of the differential gene expression in terms of a relative expression ratio allowed the paired t-test to be used to test for statistically significant differential expression between endometriosis and the autologous endometrium tissues for 76 candidate genes (see Table 1 above). Employing a combination of both the criteria of mean fold-change of 2.0 and statistically significant ΔΔC T  with P≦0.01 in the 15 cases (Table 2), 14 best candidate genes were identified, including 10 genes over-expressed by 2.0 to 5.6 folds and 4 genes under-expressed by 2.2 to 15.2 folds in endometriosis. 12 of these 14 best candidates concurred with 12 of the 14 genes identified by using the arbitrary 2-fold cutoff method. These 14 candidates were therefore chosen for further investigation.  
     Example 6  
     Identification of Preferred Genes  
      The more detailed information including gene identity and chromosome mapping on the 14 best candidates was listed in Table 3. Briefly, 10 over-expressed genes include Pim-2 oncogene (Xp11.23), Ribosomal protein L41 (12q13), Prosaposin (10q22.1), Fibulin 1 (21q13), SIPL protein (2p25.3), three uncharacterized mRNAs: L27560 (2q35), HSPC157 protein (1p36.12) and FLJ37272 (19p13.3), and two novel genes EA08 (12q15) and EA20 (2p21). 4 under-expressed genes include Distal-less homeobox 5 protein (7q21), 11 beta-hydroxysteroid dehydrogenase type 2 (16q22), protein phosphatase 2A-specific inhibitor (SET) (9q34), and ras homolog gene family, member E (RhoE) (2q23.3).  
               TABLE 3                          Summary of characteristics of the 14 best candidate genes       consistently differentially regulated in 15 paired samples                                             Gene symbol       Mean                   cDNA   (accession       fold-   P   Map   Chromosomal       clone   No.)   Gene description   change   value   locus   aberrations*                         Overexpression in endometriosis                                         EA30   LOC151361   Uncharacterized   5.6   0.0003   2q35   Gain of Chr.2           XM_098048                   (CGH, 18)       EA62   PIM2   Pim-2 oncogene   3.4   0.0002   Xp11.23           XM_010208       EA19   RPL41   Ribosomal protein   3.2   0.0017   12q13           NM_021104   L41       EA41   PSAP   Prosaposin   3.1   0.0000   10q22.1   Trisomy 10           XM_045140                   (R-banding,                               20)       EA29   FBLN1   Fibulin 1 isoform   3.0   0.0003   21q13   Gain of 21q           AF126110   D precursor               (CGH, 18)       EA08   BAC clone   Novel gene   2.7   0.0000   12q15           AC121761       EA17   HSPC157   Uncharacterized   2.4   0.0018   1p36.12           NM_014179   cDNA clone (from               CD34+ stem cells)       EA20   BAC clone   Novel gene   2.2   0.0038   2p21   Gain of Chr.2           AC073082                   (CGH, 18)       EA53   FLJ37272   Uncharacterized   2.2   0.0055   19p13.3   Tetrasomy 19           AK094591   (highly similar to               (R-banding,               GRG PROTEIN)               20)       EA44   SIPL   SIPL protein (a   2.0   0.0041   2p25.3   Gain of Chr.2           NM_018269   hepatic factor               (CGH, 18)               supporting               hepatitis C virus               replication)                 Underexpression in endometriosis                                         EA33   DLX5   Distal-less   15.2   0.0000   7q21               BC006226   homeobox 5               protein       EA60   HSD11B2   11 beta-   3.2   0.0023   16q22   Loss of 16q           NM_000196   hydroxysteroid               (CGH, 17);               dehydrogenase               Monosomy 16               type 2               (FISH, 19)       EA27   SET   A heat-stable   2.9   0.0015   9q34   Loss of 9q           NM_003011   protein               (CGH, 17)               phosphatase 2A-               specific inhibitor               (I2PP2A)       EA58   ARHE   Ras homolog gene   2.2   0.0100   2q23.3           NM_005168   family, member E                 *Common chromosome aberrations previously identified in endometriosis tissue or cell line by CGH, FISH and R-binding (see, e.g., Gogusev et al, 1999 Hum. Genet., 105: 444-451; Gogusev et al, 2000 Mol. Hum. Reprod., 6: 821-827; Shin et al, 1997 Hum. Genet., 100: 401-406; Bouquet de Joliniere et al, 1997 Hum. Reprod. Update, 3: 117-123).             
 
     EXAMPLE 7  
     Clustering of Gene Expression  
      Cluster analysis was performed to identify the specific gene groups of interest and to display the global picture of differential regulation of 76 candidate genes in 15 pairs of clinical samples. The genes and clinical samples were grouped on the basis of similarities of gene expression patterns by hierarchical clustering algorithm using the Cluster software (Eisen et al, 1998 cited above). The result was displayed by TreeView software (Eisen et al, 1998 cited above) and shown in  FIG. 5B . Based on the gene expression patterns, the 76 candidate genes were generally divided in four different groups, i.e. b1 , b2, b3 and b4 ( FIG. 5B ). The group b1 included the genes consistently over-expressed in most endometriosis samples while the genes in the group b3 were consistently under-expressed. The group b2 included most genes whose expression patterns exhibited a transition from over-expression to under-expression in endometriosis across  FIG. 4B  from the left to right. The group b4 included three immediate-early genes and one uncharacterized gene, showing a unique expression pattern that is totally different from those of the other 72 genes.  
      A zoom-in picture for the 14 best candidate genes (Table 3) were similarly displayed in  FIG. 5C  to demonstrate how these best candidate genes from the group b1 and b3 ( FIG. 5B ) were differentially regulated in each paired samples. It was clearly demonstrated that the 10 candidate genes on the top part in  FIG. 5C  were consistently over-expressed in the 15 cases while the other 4 genes on the bottom were consistently under-expressed.  
     Example 8  
     Demonstration of Clinical Co-relations  
      As shown in  FIG. 5B , based on the similarities of gene expression patterns by hierarchical clustering, the majority of cases with early disease (stage I &amp; II) clustered on the left-hand side of the figure, whereas the cases with advanced disease (stage III &amp; IV) aggregated on the right-hand side. It is noteworthy that moving across  FIG. 5B  from the left to right, the expression patterns of the genes in the group b2 exhibited a transition from over-expression to under-expression in endometriosis when compared to the paired endometrium tissue, indicating that some genes may be differentially regulated at different stages of the disease. In order to identify the genes with stage-specific regulation, statistic analyses for 76 candidate genes were performed in two groups of patients, namely, early disease (stage I &amp; II) and advanced disease (stage III &amp; IV), respectively. Employing the same criteria of mean fold-change of 2.0 and statistically significant ΔΔC T  with P≦0.01, 45 genes were differentially expressed in the paired samples in at least one group of patients. Of these 45 genes, 34 were specific to the early disease, 4 specific for the advanced disease and the remaining 7 genes were differentially expressed in both groups. The detailed information was shown in Table 4.  
               TABLE 4                          Statistic analysis of differential gene expression                                                     Mean fold       Mean fold       Mean fold           cDNA   Sequence   change at       change at stage       change in all       clone   homology   stage I &amp; II   P value   III &amp; IV   P value   (I-IV)   P value                         Overexpression in endometriosis                                             EA30   LOC151361   8.7   0.0108   4.2   0.0195   5.6   0.0003       EA62   PIM2   3.9   0.0009   3.1   0.0238   3.4   0.0002       EA19   RPL41   7.7   0.0015   1.8   0.1225   3.2   0.0017       EA41   PSAP   4.9   0.0008   2.3   0.0030   3.1   0.0000       EA29   FBLN1   5.0   0.0055   2.1   0.0205   3.0   0.0003       EA08   Novel   4.2   0.0001   2.0   0.0003   2.7   0.0000       EA17   HSPC157   4.9   0.0003   1.5   0.1549   2.4   0.0018       EA20   Novel   4.0   0.0006   1.5   0.2263   2.2   0.0038       EA53   FLJ37272   4.8   0.0009   1.3   0.3178   2.2   0.0055       EA44   SIPL   3.4   0.0012   1.4   0.2141   2.0   0.0041       EA74   FLJ22547   4.2   0.0040   −1.2   0.7114   1.6   0.1743       EA64   NBP   3.8   0.0008   1.2   0.6200   1.9   0.0283       EA57   ERH   3.7   0.0041   −1.1   0.7171   1.6   0.1605       EA42   FLJ12619   3.4   0.0020   1.2   0.3660   1.8   0.0085       EA75   FLJ33814   3.2   0.0058   1.0   0.9827   1.6   0.0740       EA14   HTR2A   3.2   0.0021   1.1   0.6222   1.7   0.0183       EA38   FLJ10416   3.1   0.0009   1.3   0.0508   1.9   0.0007       EA63   MRG15   3.1   0.0033   1.3   0.2364   1.8   0.0053       EA59   SRRM1   3.1   0.0024   1.1   0.6553   1.7   0.0265       EA21   LOC126133   3.0   0.0035   1.1   0.6367   1.7   0.0242       EA54   HNRPL   2.9   0.0024   1.0   0.9539   1.5   0.0577       EA04   MYBPC   2.8   0.0000   1.4   0.2096   1.8   0.0030       EA50   CDK2   2.8   0.0041   1.3   0.4116   1.8   0.0233       EA06   NDUFA5   2.8   0.0025   1.3   0.1868   1.7   0.0034       EA18   TSAP19   2.8   0.0097   1.0   0.9409   1.5   0.1553       EA73   Novel   2.7   0.0036   1.5   0.1061   1.9   0.0019       EA15   RPS25   2.4   0.0004   1.4   0.1348   1.7   0.0018       EA36   Adam17   2.4   0.0002   1.3   0.1679   1.7   0.0019       EA25   Ss18   2.4   0.0109   1.1   0.6457   1.5   0.0346       EA11   FLJ10952   2.2   0.0093   1.5   0.0107   1.7   0.0003       EA56   IMAGE:   2.2   0.0126   1.0   0.8324   1.3   0.1317           4856273       EA70   ARFRP1   2.2   0.0037   −1.2   0.3232   1.2   0.2734       EA69   IMAGE:   2.2   0.0043   −1.3   0.0146   1.2   0.3469           3860421       EA37   SPIN   2.2   0.0257   −1.5   0.0582   1.1   0.6988       EA51   FLJ11472   2.2   0.0165   −1.5   0.1262   1.1   0.8099       EA01   CTNNB1   2.1   0.0108   1.1   0.6785   1.5   0.0786       EA71   IMAGE:   2.1   0.0106   −1.3   0.2105   1.1   0.5708           3048642       EA13   KIAA0495   2.0   0.0040   −1.1   0.6953   1.3   0.1230                 Underexpression in endometriosis                                             EA33   DLX5   −3.7   0.0129   −38.7   0.0000   −15.2   0.0000       EA60   HSD11B2   −1.4   0.4760   −5.6   0.0010   −3.2   0.0023       EA27   SET   −3.0   0.0222   −2.9   0.0331   −2.9   0.0015       EA58   ARHE   −1.6   0.2675   −2.7   0.0248   −2.2   0.0100       EA52   COL7A1   2.0   0.0722   −5.4   0.0030   −2.1   0.0912       EA16   RPL10A   −1.3   0.2875   −2.3   0.0092   −1.8   0.0050       EA22   JUN-D   −3.3   0.0319   −1.1   0.8064   −1.7   0.1157                  
 
     Example 9  
     Differential Regulation of Several Immediate-early Genes  
      As stated above, three of four genes in the group b4 ( FIG. 5B ) are known to be the immediate-early genes, including EA22 (jun-D), EA26 (Egr-1) and EA40 (c-jun). These three genes behaved very similarly in terms of the relative expression ratio in each pair of samples (b4,  FIG. 5B ). But they exhibited very distinct expression patterns in the 15 cases. Based on the expression data of these three genes, the 15 paired samples were re-grouped by clustering and displayed in  FIG. 5D . It was clearly demonstrated that two opposite expression patterns, d1 and d3, and one transient state, d2, existed in these 15 cases. These three different patterns appear independent of the disease stages as well as the menstrual cycle (Table 2).  
      All publications cited in this specification are incorporated herein by reference herein. While the invention has been described with reference to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.