EP2118321A2 - Genemap of the human genes associated with endometriosis - Google Patents

Genemap of the human genes associated with endometriosis

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Publication number
EP2118321A2
EP2118321A2 EP08741999A EP08741999A EP2118321A2 EP 2118321 A2 EP2118321 A2 EP 2118321A2 EP 08741999 A EP08741999 A EP 08741999A EP 08741999 A EP08741999 A EP 08741999A EP 2118321 A2 EP2118321 A2 EP 2118321A2
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EP
European Patent Office
Prior art keywords
gene
tables
nucleic acid
expression
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP08741999A
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German (de)
French (fr)
Other versions
EP2118321A4 (en
Inventor
Abdelmajid Belouchi
John Verner Raelson
Bruno Paquin
Sandy Briand
Daniel Dubois
Paul Van Eerdewegh
Jonahan Segal
Randall David Little
Tim Keith
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Genizon Biosciences Inc
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Genizon Biosciences Inc
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Publication of EP2118321A2 publication Critical patent/EP2118321A2/en
Publication of EP2118321A4 publication Critical patent/EP2118321A4/en
Withdrawn legal-status Critical Current

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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the field of genomics and genetics, including genome analysis and the study of DNA variations.
  • the invention relates to the fields of pharmacogenomics, diagnostics, patient therapy and the use of genetic haplotype information to predict an individual's susceptibility to ENDOMETRIOSIS disease and/or their response to a particular drug or drugs, so that drugs tailored to genetic differences of population groups may be developed and/or administered to the appropriate population.
  • the invention also relates to a GeneMap for ENDOMETRIOSIS disease, which links variations in DNA (including both genie and non-genic regions) to an individual's susceptibility to ENDOMETRIOSIS disease and/or response to a particular drug or drugs.
  • the invention further relates to the genes disclosed in the GeneMap (see Tables 2-4 and examples of the GeneMap in the Example section herein), which is related to methods and reagents for detection of an individual's increased or decreased risk for ENDOMETRIOSIS disease and related sub-phenotypes, by identifying at least one polymorphism in one or a combination of the genes from the GeneMap. Also related are the candidate regions identified in Table 1 , which are associated with ENDOMETRIOSIS disease.
  • the invention further relates to nucleotide sequences of those genes including genomic DNA sequences, DNA sequences, single nucleotide polymorphisms (SNPs), other types of polymorphisms (insertions, deletions, microsatellites), alleles and haplotypes (see Sequence Listing and Tables 5-16).
  • SNPs single nucleotide polymorphisms
  • other types of polymorphisms insertions, deletions, microsatellites
  • alleles and haplotypes see Sequence Listing and Tables 5-16.
  • the invention further relates to isolated nucleic acids comprising these nucleotide sequences and isolated polypeptides or peptides encoded thereby. Also related are expression vectors and host cells comprising the disclosed nucleic acids or fragments thereof, as well as antibodies that bind to the encoded polypeptides or peptides.
  • the present invention further relates to ligands that modulate the activity of the disclosed genes or gene products.
  • the invention relates to diagnostics and therapeutics for ENDOMETRIOSIS disease, utilizing the disclosed nucleic acids, polymorphisms, chromosomal regions, GeneMaps, polypeptides or peptides, antibodies and/or ligands and small molecules that activate or repress relevant signaling events.
  • ENDOMETRIOSIS is defined as the presence of endometrial-like tissue growing outside the uterine cavity. It is also associated with significant impairment in quality of life for affected women due to severe pain during menstruation and sexual intercourse, and infertility.
  • the pathophysiology of ENDOMETRIOSIS remains enigmatic. As a result, current therapeutic strategies are mainly palliative and non-curative.
  • Surgery is the first-line treatment to remove ovarian endometriomas and to correct ENDOMETRIOSIS-associated adhesions that can distort pelvic anatomy. Nevertheless, patients who undergo surgical procedures have recurrence of ENDOMETRIOSIS in up to 47% of cases and recurrence of adhesions in up to 89% of cases.
  • New research treatments include the use of aromatase inhibitors together with progestin or together with oral contraceptives. However, ENDOMETRIOSIS recurs once all these treatments are stopped.
  • Medical pharmacological treatments such as the androgenic therapies, danazol and gestrinone, the constellation of GnRH agonists, buserelin, goserelin, leuprolide, nafarelin and triptorelin, GnRH antagonists, cetrorelix and abarelix, as well as the progestogens, including medroxyprogesterone acetate, induce lesion atrophy by suppressing the production of estrogen.
  • These approaches are not without unwanted side effects.
  • Danazol and gestrinone include weight gain, hirsuitism, acne, mood changes and metabolic effects on the cardiovascular system.
  • the group of GnRH agonists and antagonists are found to cause a profound suppression of estrogen leading to vasomotor effects (hot flashes) and depletion of bone mineral density, which restricts their use to only six months of therapy.
  • the group of progestogens including medroxyprogesterone acetate, suppress the gonadotropins, but do not down-regulate ovarian estrogen production to the same extent as the GnRH analogues.
  • the side effects include irregular bleeding, bloating, weight gain and metabolic effects on the cardiovascular system.
  • the present invention relates specifically to a set of ENDOMETRIOSIS disease-causing genes (GeneMap) and targets which present attractive points of therapeutic intervention and diagnostics.
  • identifying susceptibility genes associated with ENDOMETRIOSIS disease and their respective biochemical pathways will facilitate the identification of diagnostic markers as well as novel targets for improved therapeutics. It will also improve the quality of life for those afflicted by this disease and will reduce the economic costs of these afflictions at the individual and societal level.
  • the identification of those genetic markers would provide the basis for novel genetic tests and eliminate or reduce the therapeutic methods currently used.
  • the identification of those genetic markers will also provide the development of effective therapeutic intervention for the battery of laboratory, phsychological and clinical evaluations typically required to diagnose ENDOMETRIOSIS. The present invention satisfies this need.
  • filename Table1.txt, date recorded: February 6, 2008, file size: 11 kilobytes;
  • filename Table2.txt, date recorded: February 6, 2008, file size: 35 kilobytes;
  • filename Table3.txt, date recorded: February 6, 2008, file size: 278 kilobytes;
  • filename Table4.txt, date recorded: February 6, 2008, file size: 2 kilobytes;
  • filename Table5.1.txt, date recorded: February 6, 2008, file size: 81 kilobytes;
  • filename Table5.2.txt, date recorded: February 6, 2008, file size: 65 kilobytes;
  • filename Table6.1.txt, date recorded: February 6, 2008, file size: 6 kilobytes;
  • filename Table6.2.txt, date recorded: February 6, 2008, file size: 12 kilobytes;
  • filename Table7.1.txt, date recorded: February 6, 2008, file size: 15 kilobytes;
  • filename Table7.2.txt, date recorded: February 6, 2008, file size: 14 kilobytes;
  • filename Table8.1.txt, date recorded: February 6, 2008, file size: 23 kilobytes; [00025] filename: Table8.2.txt, date recorded: February 6, 2008, file size: 22 kilobytes;
  • filename Table9.1 , date recorded: February 6, 2008, file size: 4 kilobytes;
  • filename Table9.2, date recorded: February 6, 2008, file size: 3 kilobytes;
  • filename Table10.1 , date recorded: February 6, 2008, file size: 44 kilobytes;
  • filename Table10.2, date recorded: February 6, 2008, file size: 17 kilobytes;
  • filename TableH .1 , date recorded: February 6, 2008, file size: 31 kilobytes;
  • filename Table11.2, date recorded: February 6, 2008, file size: 42 kilobytes;
  • filename Table12.1 , date recorded: February 6, 2008, file size: 17 kilobytes;
  • filename Table12.2, date recorded: February 6, 2008, file size: 11 kilobytes;
  • filename Table13.1 , date recorded: February 6, 2008, file size: 27 kilobytes;
  • filename Table13.2, date recorded: February 6, 2008, file size: 16 kilobytes;
  • filename Table14.1 , date recorded: February 6, 2008, file size: 6 kilobytes; [00037] filename: Table14.2, date recorded: February 6, 2008, file size: 4 kilobytes;
  • filename Table15.1 , date recorded: February 6, 2008, file size: 13 kilobytes;
  • filename Table15.2, date recorded: February 6, 2008, file size: 8 kilobytes;
  • filename Table16.1 , date recorded: February 6, 2008, file size: 4 kilobytes;
  • filename Table16.2, date recorded: February 6, 2008, file size: 3 kilobytes;
  • filename Table17, date recorded: February 6, 2008, file size: 16 kilobytes;
  • filename Table18, date recorded: February 6, 2008, file size: 11 kilobytes.
  • Figure 1 Emerging endometriosis GeneMap.
  • Figure 2 Emerging endometriosis GeneMap with sub-phenotype.
  • FIG. 4A-G All-Stage, Whole-Body Sections throughout the embryonic (1 and 2), postnatal developmental stages (3 and 5) and adulthood (6 and 7).
  • Fig. 4H Adult Mouse Reproductive Organs: 1. Uterus, control; 2. Uterus, gestation day 5.5; 3. Uterus, gestation day 7.5; 4. Ovary; 5. Mammary gland; 6. Prostate; 7. Epididymis; 8. Testis; 9. Seminal vesicle; Fig. 41: Adult Mouse Tissue Array, General: 10. Brain, sagittal sections; 11. Thyroid; 12.
  • FIG. 5 H2AFY expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice.
  • Figs. A-D X-ray film autoradiography following hybridization with antisense riboprobe (Seq ID: 6717) after 3-day exposure, showing a pattern of H2AFY mRNA distribution seen as bright labeling on dark field.
  • Fig. E Control (sense, Seq ID: 6716) hybridization of the section comparable to D.
  • FIG. 6A Anatomical view of the adult mouse after staining with cresyl violet.
  • Fig. 6B X-ray film autoradiography after hybridization with antisense (Seq ID: 6717) riboprobe showing the presence of H2AFY mRNA in the brain, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination.
  • Fig. 6C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B.
  • BM - bone marrow Br - brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; LN - lymph node; Lu - lung; Ri - ribs; SG - salivary gland; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense.
  • FIG. 7 A X-ray film autoradiography after hybridization with antisense (Seq ID: 6717) riboprobe showing H2AFY mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Expression sites are evident in the ovary, control uterus and uterine tissue at gestation stages day 5.5 and 7.5.
  • Fig. 7 B H2AFY mRNA shown in the general tissue array (TA). Low to medium levels of expression are evident in most tissue including the brain, pituitary gland, adrenal gland, thyroid, testis, splee, kidney and prostate.
  • Fig. 7 C H2AFY mRNA in the brain tissue arrays; expression is evident in the olfactory lobe, hippocampus, hypothalamus and cerebellum.
  • Fig. 7 D Control (sense, Seq ID: 6716) hybridization of the section comparable to B.
  • Adr - adrenal gland Br - brain; Hip - hippocampus; Cb - cerebellum; Hy - hypothalamus; K - kidney; Li - liver; Lu - lung; OL - olfactory lobe; Ov - ovary; Pit - pituitary gland; Pr - prostate; SG - salivary gland; Sp - spleen; Td - thyroid gland; Te - testis; Th - thymus; UB - urinary bladder; Ut - uterus; UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense.
  • FIG. 8A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the hippocampus seen as bright on darkfield illumination.
  • Fig. 8B Fragment of the hippocampus with H2AFY mRNA labeled area CA1 neurons (arrow)
  • Fig. 8C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B.
  • Fig. 8A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the hippocampus seen as bright on darkfield illumination.
  • Fig. 8B Fragment of the hippocampus with H2AFY mRNA labeled area CA1 neurons (arrow)
  • Fig. 8C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable
  • FIG. 9 H2AFY expression in the adult mouse brain bone marrow and dorsal root ganglion.
  • Fig. 9A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the bone marrow region and dorsal root ganglion seen as bright on darkfield illumination.
  • Fig. 9B Fragment of the bone marrow with H2FY mRNA labeled cells (arrow), bone unlabeled, at higher magnification.
  • Fig. 9C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B.
  • Fig. 9A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the bone marrow region and dorsal root ganglion seen as bright on darkfield illumination.
  • FIG. 10 H2AFY expression in the thymus.
  • Fig. 10A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA as bright labeling under darkfield illumination.
  • Fig. 10B At higher magnification, it is seen that H2AFY mRNA labeling follow the cell density which is higher in the cortex and lower in the medulla.
  • Fig. 10C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to A. Abbreviations: Cx - cortex; Me - medulla; (s) - sense. Magnifications: (A) x 25; (B and C) x 405.
  • FIG. 11A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA seen as bright labeling under darkfield illumination mostly in the corpus luteum.
  • Fig. 11 B At higher magnification H2AFY mRNA labeling is seen in the corpus luteum cells and in the follicular cells (arrows). Theca cells seem to not express H2AFY.
  • Fig. 11C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Abbreviations: CL - corpus luteum; FC - follicular cells; T - theca; (s) - sense. Magnifications: (A) x 25; (B and C) x 405.
  • FIG. 12A Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the endometrium epithelial cells layer (arrow) seen as bright under darkfield illumination.
  • Fig. 12B The same section seen at lightfield illumination and cresyl violet staining.
  • Fig. 12C Fragment of the uterine epithelium, labeled (arrow) at high magnification.
  • Fig. 12D Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to A at darkfield illumination.
  • FIG. 12E The same section seen at lightfield illumination and cresyl violet staining.
  • Fig. 12F Fragment of the uterine epithelium following control (sense, Seq ID: 6716) hybridization.
  • FIG. 13 H2AFY expression in the female uterus 7,5 days pregnant.
  • Fig. 13A Emulsion autoradiography, after hybridization with antisense (Seq ID: 6717) riboprobe, throughout the peripheral region of the uterus. H2AFY mRNA labeling is present in mostly endometrium cells and much less in the myometrium.
  • Fig. 13B Centrally located deciduas with labeled giant cells originated from the ectoplacental cone (heavy arrows) and the presumptive trophoblasts of trophectoderm origin (small arrows).
  • Fig. 13C Control (sense, SEQIDPROBE2]) hybridization of an adjacent section comparable to B. Abbreviations: BV - blood vessels; E - endometrium; - H - hondrion; M - myometrium; (s) - sense. Magnification: x 380.
  • FIG. 14A Emulsion autoradiography, after hybridization with antisense (Seq ID: 6717) riboprobe, throughout the testis showing H2AFY mRNA labeling as bright under lightfield illumination. Labeling is present in a proportion of seminiferous tubules (arrow).
  • Fig. 14B Fragment of the seminiferous tubule showing H2AFY mRNA labeling concentrated mostly in the layer of spermatogonia and much less in spermatocyte layer. There is no detectable labeling in the spermatozoa.
  • Fig. 14C Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B.
  • Figure 15 MAD2L2 expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice.
  • Figs. 15A-D X-ray film autoradiography following hybridization with antisense (Seq ID: 6719) riboprobe after 4-day exposure, showing a pattern of MAD2L2 mRNA distribution seen as bright labeling on dark field.
  • Fig. 15E Control (sense, Seq ID: 6718) hybridization of the section comparable to D.
  • FIG.16A Anatomical view of the adult mouse after staining with cresyl violet.
  • Fig.16B X-ray film autoradiography following hybridization with antisense (Seq ID: 6719) riboprobe showing the presence of MAD2L2 mRNA in the salivary gland, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination.
  • Fig.16C Control (sense, Seq ID: 6718) hybridization of an adjacent section comparable to Fig.16B.
  • FIG. 17A MAD2L2 expression in the adult mouse tissue arrays.
  • FIG. 17A X-ray film autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. High expression sites are evident in the ovary and testis and in the uterine tissue at gestation stages day 5.5 and 7.5.
  • Fig. 17B MAD2L2 mRNA shown in the general tissue array (TA). Low to medium level expression levels are evident in most tissue including brain, adrenal gland, spleen, thymus and liver. High MAD2L2 expression levels are confirmed in the testis and ovary.
  • TA general tissue array
  • FIG. 18A Emulsion autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA labeling in the wall of the seminiferous tubules seen as dark silver grains under lightfield illumination; cresyl violet staining of cell nuclei. By topography, the labeled cells may be identified a spermatocytes. Spermatogonia and spermatozoa appears as unlabeled. There is no labeling in the interstitial space Leydig cells.
  • Fig. 18B Control (sense, Seq ID: 6718) hybridization of an adjacent section comparable to A.
  • FIG. 19 MAD2L2 expression in the pregnant female uterus.
  • Fig. 19A Emulsion autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA labeling in the uterus on day 7.5 post coitum. Silver labeling is seen as dark under lightfield illumination; cresyl violet staining of cell nuclei. By topography, the labeled cells may be identified as endometrial cells. Peripherally located myometrium seems to be free of labeling.
  • Fig. 19B MAD2L2 labeling in the endometrium region with high PCNA activity (not shown).
  • FIG. 20 MCM3AP expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice.
  • Figs. 20A-D X-ray film autoradiography following hybridization with antisense riboprobe (Seq ID: 6721) after 5-day exposure, showing a pattern of MCM3AP mRNA distribution seen as bright labeling on darkfield.
  • Fig. 20E Control (sense, Seq ID: 6720) hybridization of the section comparable to D.
  • FIG. 21 MCM3AP expression in the adult mouse.
  • Fig. 21A Anatomical view of the adult mouse after staining with cresyl violet.
  • Fig. 21 B X- ray film autoradiography after hybridization with antisense riboprobe (Seq ID: 6721) showing the presence of MCM3AP mRNA in the brain, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination.
  • Fig. 21C Control (sense, Seq ID: 6720) hybridization of an adjacent section comparable to B.
  • BM - bone marrow Br - brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; LN - lymph node; Lu - lung; SG - salivary gland; SI - small intestine; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense.
  • FIG. 22 MCM3AP expression in the adult mouse tissue arrays.
  • Fig. 22A X-ray film autoradiography, after hybridization with antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Overall low mRNA concentration is evident.
  • Fig. 22B MCM3AP mRNA shown in the general tissue array (TA). MCM3AP expression levels are at the limit of the detection by ISH in most tissues including the brain, trigeminal ganglion, adrenal gland and spleen. Slightly elevated mRNA concentrations occur in the thymus.
  • Fig. 22A X-ray film autoradiography, after hybridization with antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Overall low mRNA concentration is evident.
  • Fig. 22B MCM3AP m
  • Adr - adrenal gland Adr - adrenal gland; Br - brain; Hip - hippocampus; Cb - cerebellum; OL - olfactory lobe; Ov - ovary; SG - salivary gland; Sp - spleen; Te - testis; Th - thymus; Ut - uterus; UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense.
  • FIG. 23 MCM3AP expression in the adult mouse brain hippocampus and cerebellum.
  • Fig. 23A Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA labeling (arrow) in the hippocampus area CA1 seen as bright labeling under darkfield illumination.
  • Fig. 23B The same fragment of the hippocampus seen under brightfield illumination. Staining tissue with cresyl violet reveals a high density of labeled cell layer.
  • Fig. 23C Control (sense, Seq ID: 6720) hybridization of an adjacent section comparable to A under darkfield.
  • Fig. 23D The same section under brightfield illumination.
  • FIG. 24 NRXN1 expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice.
  • Figs. 24A-D X-ray film autoradiography following hybridization with antisense Seq ID: 6723 riboprobe after 2-day exposure, showing a pattern of NRXN1 mRNA distribution seen as bright labeling on dark field.
  • Fig. 24E Control (sense, Seq ID: 6722) hybridization of the section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; DRG - dorsal root ganglia; SC - spinal cord; (s) - sense. Magnification x 1.6.
  • FIG. 25 NRXN 1 expression in the adult mouse.
  • Fig. 25A Anatomical view of the adult mouse after staining with cresyl violet.
  • Fig. 25B X-ray film autoradiography after hybridization with antisense riboprobe (Seq ID: 6723) showing the presence of NRXN1 mRNA in the brain, spinal cord, dorsal root ganglia and trigeminal ganglion.
  • Fig. 25C Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to B.
  • FIG. 26 NRXN1 expression in the adult mouse tissue arrays.
  • Fig. 26A Two-day X-ray film autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Overall low mRNA concentration is evident.
  • Fig. 26B NRXN 1 mRNA shown in the general tissue array (TA). NRXN 1 expression is detectable in the CNS (brain), PNS (trigeminal ganglion) and endocrine glands (pituitary and adrenals).
  • Fig. 26C NRXN 1 mRNA in the brain tissue arrays.
  • Fig. 26D Control (sense, Seq ID: 6722) hybridization of the section comparable to B.
  • Figure 27 NRXN1 expression in the adult mouse CNS hippocampus, cortex and PNS trigeminal ganglion.
  • Fig. 27A Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the cortex and hippocampus area CA1 seen as bright on darkfield illumination. Note strongly labeled deep cortical sub layer VIb.
  • Fig. 27B Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination.
  • Fig. 27C Fragment of the trigeminal ganglion seen under brightfield illumination.
  • Fig. 27A Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the cortex and hippocampus area CA1 seen as bright on darkfield illumination. Note strongly labeled deep cortical sub layer VIb.
  • FIG. 27D Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to C.
  • Fig. 27E Cerebral cortex at higher magnification. Large arrow indicates a labeled neuron. Thin arrow points an unlabeled presumptive glial cell.
  • Fig. 27F Trigeminal ganglion at higher magnification. Large arrows indicate the sensory neurons, labeled. Thin arrows point the unlabeled satellite cells. Schwann cell seen in the nerve tissue appear unlabeled. Magnifications: (A to D) x 60; (E and F) x 250.
  • FIG. 28 NRXN1 expression in the newborn (p1) mouse PNS sensory dorsal root ganglion and ortosympathetic paravertebral ganglion.
  • Fig. 28A Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the dorsal root ganglion and paravertebral ganglion on darkfield illumination.
  • Fig. 28B The same section seen under brightfield illumination.
  • Fig. 28C Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination.
  • D The same section seen under brightfield illumination.
  • FIG. 29 NRXN 1 expression in the postnatal and adult mouse PNS visceral Auerbach plexus.
  • Fig. 29A Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the intestine of p10 mouse. Arrows indicate group of neurons present in the smooth muscle cell layer.
  • Fig. 29B The same section seen under brightfield illumination.
  • Fig. 29C Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination.
  • Fig. 29D The same section seen under brightfield illumination.
  • NRXN1 mRNA-labeled neuron in the Auerbach plexus (arrows) in the postnatal mouse intestine found in the space between circular and longitudinal smooth muscles layer showing .
  • Fig. 29F NRXN1 mRNA-labeled neuron in Auerbach plexus in the adult mouse intestine showing an inferior labeling intensity when compared to that of p10 mouse plexus.
  • Table 1 List of Endometriosis disease candidate regions identified from the Genome Wide Scan association analyses.
  • the first column denotes the region identifier.
  • the second and third columns correspond to the chromosome and cytogenetic band, respectively.
  • the fourth and fifth columns correspond to the chromosomal start and end coordinates of the NCBI genome assembly derived from build 36.
  • Table 2 List of candidate genes from the regions identified from the genome wide association analysis.
  • the first column corresponds to the region identifier provided in Table 1.
  • the second and third columns correspond to the chromosome and cytogenetic band, respectively.
  • the fourth and fifth columns corresponds to the chromosomal start coordinates of the NCBI genome assembly derived from build 36 (B36) and the end coordinates (the start and end position relate to the + orientation of the NCBI assembly and don't necessarily correspond to the orientation of the gene).
  • the sixth and seventh columns correspond to the official gene symbol and gene name, respectively, and were obtained from the NCBI Entrez Gene database.
  • the eighth column corresponds to the NCBI Entrez Gene Identifier (GenelD).
  • the ninth and tenth columns correspond to the Sequence IDs from nucleotide (cDNA) and protein entries in the Sequence Listing.
  • Table 3 List of candidate genes based on EST clustering from the regions identified from the various genome wide analyses.
  • the first column corresponds to the region identifier provided in Table 1.
  • the second column corresponds to the chromosome number.
  • the third and fourth columns correspond to the chromosomal start and end coordinates of the NCBI genome assemblies derived from build 36 (B36).
  • the fifth column corresponds to the ECGene Identifier, corresponding to the ECGene track of UCSC. These ECGene entries were determined by their overlap with the regions from Table 1 , based on the start and end coordinates of both Region and ECGene identifiers.
  • the sixth and seventh columns correspond to the Sequence IDs from nucleotide and protein entries in the Sequence Listing.
  • Table 4 List of micro RNA (miRNA) from the regions identified from the genome wide association analyses derived from build 36 (B36). To identify the miRNA from B36, these miRNA entries were determined by their overlap with the regions from Table 1 , based on the start and end coordinates of both Region and miRNA identifiers. The first column corresponds to the region identifier provided in Table 1. The second column corresponds to the chromosome number. The third and fourth columns correspond to the chromosomal start and end coordinates of the NCBI genome assembly derived from build 36 (the start and end position relate to the + orientation of the NCBI assembly and do not necessarily correspond to the orientation of the miRNA).
  • miRNA micro RNA
  • the fifth and sixth columns correspond to the miRNA accession and miRNA id, respectively, and were obtained from the miRBase database.
  • the seventh column corresponds to the NCBI Entrez Gene Identifier (GenelD).
  • the eighth column corresponds to the Sequence ID from nucleotide (RNA) in the Sequence Listing.
  • Table 5 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 5.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 6.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 6.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 7.1 Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: NOT INFERTILE. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - log 10 P values for GWS, - log 10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
  • GWS genome wide scan
  • Table 7.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 7.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 8.1 Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: NOT PELVIC PAIN. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
  • GWS genome wide scan
  • Table 8.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 8.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 9.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 9.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 10.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 10.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 11.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 11.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data has PRKCE- 1-1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
  • Table 12.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 12.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 13.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 13.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 14.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 14.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data has SYNE1-1 cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
  • Table 15.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 15.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 16.2 List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 16.1.
  • the first column lists the region ID as presented in Table 1.
  • the Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported.
  • the Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column.
  • the Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question.
  • the RR column gives to the relative risk for each particular haplotype.
  • the remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker.
  • the Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
  • Table 17 Description of primer sequences used for the semiquantitative gene expression profiling by RT-PCR (see Example section for details).
  • Allele One of a pair, or series, of forms of a gene or non-genic region that occur at a given locus in a chromosome. Alleles are symbolized with the same basic symbol (e.g., B for dominant and b for recessive; B1 , B2, Bn for n additive alleles at a locus). In a normal diploid cell there are two alleles of any one gene (one from each parent), which occupy the same relative position (locus) on homologous chromosomes. Within a population there may be more than two alleles of a gene. See multiple alleles. SNPs also have alleles, i.e., the two (or more) nucleotides that characterize the SNP.
  • Amplification of nucleic acids refers to methods such as polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known in the art and are described, for example, in U.S. Patent Nos. 4,683,195 and 4,683,202. Reagents and hardware for conducting PCR are commercially available. Primers useful for amplifying sequences from the disorder region are preferably complementary to, and preferably hybridize specifically to, sequences in the disorder region or in regions that flank a target region therein. Genes from Tables 2-4 generated by amplification may be sequenced directly. Alternatively, the amplified sequence(s) may be cloned prior to sequence analysis.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Antigenic component is a moiety that binds to its specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
  • Antibodies refer to polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, that can bind to proteins and fragments thereof or to nucleic acid sequences from the disorder region, particularly from the disorder gene products or a portion thereof.
  • the term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Proteins may be prepared synthetically in a protein synthesizer and coupled to a carrier molecule and injected over several months into rabbits. Rabbit sera are tested for immunoreactivity to the protein or fragment.
  • Monoclonal antibodies may be made by injecting mice with the proteins, or fragments thereof.
  • Monoclonal antibodies can be screened by ELISA and tested for specific immunoreactivity with protein or fragments thereof (Harlow et al. 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). These antibodies will be useful in developing assays as well as therapeutics.
  • Associated allele refers to an allele at a polymorphic locus that is associated with a particular phenotype of interest, e.g., a predisposition to a disorder or a particular drug response.
  • cDNA refers to complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, included in a cloning vector or PCR amplified. This term includes genes from which the intervening sequences have been removed.
  • cDNA library refers to a collection of recombinant DNA molecules containing cDNA inserts that together comprise essentially all of the expressed genes of an organism or tissue.
  • a cDNA library can be prepared by methods known to one skilled in the art (see, e.g., Cowell and Austin, 1997, "DNA Library Protocols," Methods in Molecular Biology). Generally, RNA is first isolated from the cells of the desired organism, and the RNA is used to prepare cDNA molecules.
  • Cloning refers to the use of recombinant DNA techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to use methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • Cloning vector refers to a plasmid or phage DNA or other DNA molecule that is able to replicate in a host cell.
  • the cloning vector is typically characterized by one or more endonuclease recognition sites at which such DNA sequences may be cleaved in a determinable fashion without loss of an essential biological function of the DNA, and which may contain a selectable marker suitable for use in the identification of cells containing the vector.
  • Coding sequence or a protein-coding sequence is a polynucleotide sequence capable of being transcribed into mRNA and/or capable of being translated into a polypeptide or peptide.
  • the boundaries of the coding sequence are typically determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • Complement of a nucleic acid sequence refers to the antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
  • Disorder region refers to the portions of the human chromosomes displayed in Table 1 bounded by the markers from Tables 2-16.
  • Disorder-associated nucleic acid or polypeptide sequence refers to a nucleic acid sequence that maps to region of Table 1 or the polypeptides encoded therein (Tables 2-4, nucleic acids, and polypeptides).
  • nucleic acids this encompasses sequences that are identical or complementary to the gene sequences from Tables 2-4, as well as sequence-conservative, function- conservative, and non-conservative variants thereof.
  • polypeptides this encompasses sequences that are identical to the polypeptide, as well as function-conservative and non-conservative variants thereof.
  • alleles of naturally-occurring polymorphisms causative of ENDOMETRIOSIS disease such as, but not limited to, alleles that cause altered expression of genes of Tables 2-4 and alleles that cause altered protein levels or stability (e.g., decreased levels, increased levels, expression in an inappropriate tissue type, increased stability, and decreased stability).
  • Expression vector refers to a vehicle or plasmid that is capable of expressing a gene that has been cloned into it, after transformation or integration in a host cell.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) a regulatory sequence.
  • Function-conservative variants are those in which a change in one or more nucleotides in a given codon position results in a polypeptide sequence in which a given amino acid residue in the polypeptide has been replaced by a conservative amino acid substitution. Function-conservative variants also include analogs of a given polypeptide and any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide.
  • Founder population Also a population isolate, this is a large number of people who have mostly descended, in genetic isolation from other populations, from a much smaller number of people who lived many generations ago.
  • Gene refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein.
  • the term "gene” also refers to a DNA sequence that encodes an RNA product.
  • the term gene as used herein with reference to genomic DNA includes intervening, non-coding regions, as well as regulatory regions, and can include 5' and 3' ends.
  • a gene sequence is wild-type if such sequence is usually found in individuals unaffected by the disorder or condition of interest. However, environmental factors and other genes can also play an important role in the ultimate determination of the disorder. In the context of complex disorders involving multiple genes (oligogenic disorder), the wild type, or normal sequence can also be associated with a measurable risk or susceptibility, receiving its reference status based on its frequency in the general population.
  • GeneMaps are defined as groups of gene(s) that are directly or indirectly involved in at least one phenotype of a disorder (some non-limiting example of GeneMaps comprises varius combinations of genes from Tables 2-4). As such, GeneMaps enable the development of synergistic diagnostic products, creating "theranostics”.
  • Genotype Set of alleles at a specified locus or loci.
  • Haplotype The allelic pattern of a group of (usually contiguous) DNA markers or other polymorphic loci along an individual chromosome or double helical DNA segment. Haplotypes identify individual chromosomes or chromosome segments. The presence of shared haplotype patterns among a group of individuals implies that the locus defined by the haplotype has been inherited, identical by descent (IBD), from a common ancestor. Detection of identical by descent haplotypes is the basis of linkage disequilibrium (LD) mapping. Haplotypes are broken down through the generations by recombination and mutation.
  • IBD identical by descent
  • Detection of identical by descent haplotypes is the basis of linkage disequilibrium (LD) mapping. Haplotypes are broken down through the generations by recombination and mutation.
  • a specific allele or haplotype may be associated with susceptibility to a disorder or condition of interest, e.g., ENDOMETRIOSIS disease.
  • an allele or haplotype may be associated with a decrease in susceptibility to a disorder or condition of interest, i.e., a protective sequence.
  • Host includes prokaryotes and eukaryotes.
  • the term includes an organism or cell that is the recipient of an expression vector (e.g., autonomously replicating or integrating vector).
  • Hybridizable nucleic acids are hybridizable to each other when at least one strand of the nucleic acid can anneal to another nucleic acid strand under defined stringency conditions.
  • hybridization requires that the two nucleic acids contain at least 10 substantially complementary nucleotides; depending on the stringency of hybridization, however, mismatches may be tolerated.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, and can be determined in accordance with the methods described herein.
  • IBD Identity by descent
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity can be readily calculated by known methods, including but not limited to those described in A.M. Lesk (ed), 1988, Computational Molecular Biology, Oxford University Press, NY; D.W. Smith (ed), 1993, Biocomputing. Informatics and Genome Projects, Academic Press, NY; A.M. Griffin and H. G. Griffin, H.
  • Immunogenic component is a moiety that is capable of eliciting a humoral and/or cellular immune response in a host animal.
  • Isolated nucleic acids are nucleic acids separated away from other components (e.g., DNA, RNA, and protein) with which they are associated (e.g., as obtained from cells, chemical synthesis systems, or phage or nucleic acid libraries). Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components. In accordance with the present invention, isolated nucleic acids can be obtained by methods described herein, or other established methods, including isolation from natural sources (e.g., cells, tissues, or organs), chemical synthesis, recombinant methods, combinations of recombinant and chemical methods, and library screening methods.
  • natural sources e.g., cells, tissues, or organs
  • chemical synthesis e.g., recombinant methods, combinations of recombinant and chemical methods, and library screening methods.
  • Isolated polypeptides or peptides are those that are separated from other components (e.g., DNA, RNA, and other polypeptides or peptides) with which they are associated (e.g., as obtained from cells, translation systems, or chemical synthesis systems).
  • isolated polypeptides or peptides are at least 10% pure; more preferably, 80% or 90% pure.
  • Isolated polypeptides and peptides include those obtained by methods described herein, or other established methods, including isolation from natural sources (e.g., cells, tissues, or organs), chemical synthesis, recombinant methods, or combinations of recombinant and chemical methods.
  • Proteins or polypeptides referred to herein as recombinant are proteins or polypeptides produced by the expression of recombinant nucleic acids.
  • a portion as used herein with regard to a protein or polypeptide refers to fragments of that protein or polypeptide. The fragments can range in size from 5 amino acid residues to all but one residue of the entire protein sequence. Thus, a portion or fragment can be at least 5, 5-50, 50-100, I00-200, 200-400, 400-800, or more consecutive amino acid residues of a protein or polypeptide.
  • LD Linkage disequilibrium
  • Markers that are in high LD can be assumed to be located near each other and a marker or haplotype that is in high LD with a genetic trait can be assumed to be located near the gene that affects that trait.
  • the physical proximity of markers can be measured in family studies where it is called linkage or in population studies where it is called linkage disequilibrium.
  • LD mapping population based gene mapping, which locates disorder genes by identifying regions of the genome where haplotypes or marker variation patterns are shared statistically more frequently among disorder patients compared to healthy controls. This method is based upon the assumption that many of the patients will have inherited an allele associated with the disorder from a common ancestor (IBD), and that this allele will be in LD with the disorder gene.
  • IBD common ancestor
  • Locus a specific position along a chromosome or DNA sequence. Depending upon context, a locus could be a gene, a marker, a chromosomal band or a specific sequence of one or more nucleotides.
  • Minor allele frequency (MAF) the population frequency of one of the alleles for a given polymorphism, which is equal or less than 50%. The sum of the MAF and the Major allele frequency equals one.
  • Markers an identifiable DNA sequence that is variable (polymorphic) for different individuals within a population. These sequences facilitate the study of inheritance of a trait or a gene. Such markers are used in mapping the order of genes along chromosomes and in following the inheritance of particular genes; genes closely linked to the marker or in LD with the marker will generally be inherited with it. Two types of markers are commonly used in genetic analysis, microsatellites and SNPs.
  • Microsatellite DNA of eukaryotic cells comprising a repetitive, short sequence of DNA that is present as tandem repeats and in highly variable copy number, flanked by sequences unique to that locus.
  • Mutant sequence if it differs from one or more wild-type sequences.
  • a nucleic acid from a gene listed in Tables 2-4 containing a particular allele of a single nucleotide polymorphism may be a mutant sequence.
  • the individual carrying this allele has increased susceptibility toward the disorder or condition of interest.
  • the mutant sequence might also refer to an allele that decreases the susceptibility toward a disorder or condition of interest and thus acts in a protective manner.
  • the term mutation may also be used to describe a specific allele of a polymorphic locus.
  • Non-conservative variants are those in which a change in one or more nucleotides in a given codon position results in a polypeptide sequence in which a given amino acid residue in a polypeptide has been replaced by a non- conservative amino acid substitution.
  • Non-conservative variants also include polypeptides comprising non-conservative amino acid substitutions.
  • Nucleic acid or polynucleotide purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo polydeoxyribonucleotides. This includes single-and double- stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as protein nucleic acids (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
  • PNA protein nucleic acids
  • Nucleotide a nucleotide, the unit of a DNA molecule, is composed of a base, a 2'-deoxyribose and phosphate ester(s) attached at the 5' carbon of the deoxyribose.
  • the nucleotide needs to possess three phosphate esters but it is converted into a monoester in the process.
  • Operably linked means that the promoter controls the initiation of expression of the gene.
  • a promoter is operabiy linked to a sequence of proximal DNA if upon introduction into a host cell the promoter determines the transcription of the proximal DNA sequence(s) into one or more species of RNA.
  • a promoter is operably linked to a DNA sequence if the promoter is capable of initiating transcription of that DNA sequence.
  • Ortholog denotes a gene or polypeptide obtained from one species that has homology to an analogous gene or polypeptide from a different species.
  • Paralog denotes a gene or polypeptide obtained from a given species that has homology to a distinct gene or polypeptide from that same species.
  • Phenotype any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to, a disorder.
  • Polymorphism occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals at a single locus.
  • a polymorphic site thus refers specifically to the locus at which the variation occurs.
  • an individual carrying a particular allele of a polymorphism has an increased or decreased susceptibility toward a disorder or condition of interest.
  • a portion as used with regard to a nucleic acid or polynucleotide refers to fragments of that nucleic acid or polynucleotide.
  • the fragments can range in size from 8 nucleotides to all but one nucleotide of the entire gene sequence.
  • the fragments are at least about 8 to about 10 nucleotides in length; at least about 12 nucleotides in length; at least about 15 to about 20 nucleotides in length; at least about 25 nucleotides in length; or at least about 35 to about 55 nucleotides in length.
  • Probe or primer refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target region of a nucleic acid due to complementarity of the probe or primer sequence to at least one portion of the target region sequence.
  • Protein and polypeptide are synonymous. Peptides are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity (e.g., proteolysis, adhesion, fusion, antigenic, or intracellular activity) as the complete polypeptide sequence.
  • functional activity e.g., proteolysis, adhesion, fusion, antigenic, or intracellular activity
  • Recombinant nucleic acids nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. Portions of recombinant nucleic acids which code for polypeptides can be identified and isolated by, for example, the method of M. Jasin et al., U.S. Patent No. 4,952,501.
  • Regulatory sequence refers to a nucleic acid sequence that controls or regulates expression of structural genes when operably linked to those genes. These include, for example, the lac systems, the trp system, major operator and promoter regions of the phage lambda, the control region of fd coat protein and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells. Regulatory sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host, and may contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements and/or translational initiation and termination sites.
  • Sample refers to a biological sample, such as, for example, tissue or fluid isolated from an individual or animal (including, without limitation, plasma, serum, cerebrospinal fluid, lymph, tears, nails, hair, saliva, milk, pus, and tissue exudates and secretions) or from in vitro cell culture- constituents, as well as samples obtained from, for example, a laboratory procedure.
  • tissue or fluid isolated from an individual or animal (including, without limitation, plasma, serum, cerebrospinal fluid, lymph, tears, nails, hair, saliva, milk, pus, and tissue exudates and secretions) or from in vitro cell culture- constituents, as well as samples obtained from, for example, a laboratory procedure.
  • SNP Single nucleotide polymorphism
  • SNPs are biallelic markers although tri- and tetra-allelic markers also exist.
  • SNP A ⁇ C may comprise allele C or allele A (Tables 5-16).
  • a nucleic acid molecule comprising SNP A ⁇ C may include a C or A at the polymorphic position.
  • an ambiguity code is used in Tables 5-16 and the sequence listing, to represent the variations.
  • haplotype is used, e.g.
  • haplotype is used to describe a combination of SNP alleles, e.g., the alleles of the SNPs found together on a single DNA molecule.
  • the SNPs in a haplotype are in linkage disequilibrium with one another.
  • Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position (i.e., silent mutation).
  • Substantially homologous a nucleic acid or fragment thereof is substantially homologous to another if, when optimally aligned (with appropriate nucleotide insertions and/or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least 60% of the nucleotide bases, usually at least 70%, more usually at least 80%, preferably at least 90%, and more preferably at least 95-98% of the nucleotide bases.
  • substantial homology exists when a nucleic acid or fragment thereof will hybridize, under selective hybridization conditions, to another nucleic acid (or a complementary strand thereof). Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs.
  • selective hybridization will occur when there is at least about 55% sequence identity over a stretch of at least about nine or more nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% (M. Kanehisa, 1984, NucL Acids Res. 11 :203-213).
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least 14 nucleotides, usually at least 20 nucleotides, more usually at least 24 nucleotides, typically at least 28 nucleotides, more typically at least 32 nucleotides, and preferably at least 36 or more nucleotides.
  • Wild-type gene from Tables 2-4 refers to the reference sequence.
  • the wild-type gene sequences from Tables 2-4 used to identify the variants (polymorphisms, alleles, and haplotypes) described in detail herein.
  • the present invention is based on the discovery of genes associated with ENDOMETRIOSIS disease.
  • disease- associated loci candidate regions; Table 1 are identified by the statistically significant differences in allele or haplotype frequencies between the cases and the controls.
  • candidate regions are identified in Table 1.
  • the invention provides a method for the discovery of genes associated with ENDOMETRIOSIS disease and the construction of a GeneMap for ENDOMETRIOSIS disease in a human population, comprising the following steps (see also Example section herein).
  • the GeneMaps of the invention, presented in the Example section is provided for clarity purposes and other GeneMaps with various other combinations of genes from Tables 2-4 and/or other genes involved in the related networks or pathways are obtained by the methods of the invention:
  • Step 1 Recruit patients (cases) and controls
  • more or less than 500 patients and controls are recruited.
  • 500 patients diagnosed for ENDOMETRIOSIS disease along with two family members are recruited from the Quebec Founder Population (QFP).
  • the preferred trios recruited are parent- parent-child (PPC) trios.
  • Trios can also be recruited as parent-child-child (PCC) trios.
  • more or less than 500 trios are recruited
  • the present invention is performed as a whole or partially with DNA samples from individuals of another founder population than the Quebec population or from the general population.
  • Step 2 DNA extraction and quantitation
  • any sample comprising cells or nucleic acids from patients or controls may be used.
  • Preferred samples are those easily obtained from the patient or control.
  • Such samples include, but are not limited to blood, peripheral lymphocytes, buccal swabs, epithelial cell swabs, nails, hair, bronchoalveolar lavage fluid, sputum, or other body fluid or tissue obtained from an individual.
  • DNA is extracted from such samples in the quantity and quality necessary to perform the invention using conventional DNA extraction and quantitation techniques.
  • the present invention is not linked to any DNA extraction or quantitation platform in particular.
  • Step 3 Genotype the recruited individuals
  • assay-specific and/or locus-specific and/or allele-specific oligonucleotides for every SNP marker of the present invention are organized onto one or more arrays.
  • the genotype at each SNP locus is revealed by hybridizing short PCR fragments comprising each SNP locus onto these arrays.
  • the arrays permit a high-throughput genome wide association study using DNA samples from individuals of the Quebec founder population.
  • Such assay-specific and/or locus-specific and/or allele-specific oligonucleotides necessary for scoring each SNP of the present invention are preferably organized onto a solid support.
  • Such supports can be arrayed on wafers, glass slides, beads or any other type of solid support.
  • the assay-specific and/or locus-specific and/or allele-specific oligonucleotides are not organized onto a solid support but are still used as a whole, in panels or one by one.
  • the present invention is therefore not linked to any genotyping platform in particular.
  • one or more portions of the SNP maps are used to screen the whole genome, a subset of chromosomes, a chromosome, a subset of genomic regions or a single genomic region.
  • the individuals composing the cases and controls or the trios are preferably individually genotyped with at least 80,000 markers, generating at least a few million genotypes; more preferably, at least a hundred million.
  • individuals are pooled in cases and control pools for genotyping and genetic analysis.
  • Step 4 Exclude the markers that did not pass the quality control of the assay.
  • the quality controls comprises, but are not limited to, the following criteria: eliminate SNPs that had a high rate of Mendelian errors (cut-off at 1 % Mendelian error rate), that deviate from the Hardy-Weinberg equilibrium, that are non-polymorphic in the Quebec founder population or have too many missing data (cut-off at 1 % missing values or higher), or simply because they are non-polymorphic in the Quebec founder population (cut-off at 1 % ⁇ 10% minor allele frequency (MAF)).
  • Step 5 Perform the genetic analysis on the results obtained using haplotype information as well as single-marker association.
  • genetic analysis is performed on a subset of markers from Step 3 or from markers that passed the quality controls from Step 4.
  • the genetic analysis consists of, but is not limited to features corresponding to Phase information and haplotype structures.
  • Phase information and haplotype structures are preferably deduced from trio genotypes using Phasefinder. Since chromosomal assignment (phase) cannot be estimated when all trio members are heterozygous, an Expectation-Maximization (EM) algorithm may be used to resolve chromosomal assignment ambiguities after Phasefinder.
  • EM Expectation-Maximization
  • the PL-EM algorithm Partition-Ligation EM; Niu et a/.., Am. J. Hum. Genet. 70:157 (2002)
  • haplotypes from the "genotype" data as a measured estimate of the reference allele frequency of a SNP in 15-marker windows that advance in increments of one marker across the data set.
  • the results from such algorithms are converted into 15-marker haplotype files.
  • the individual 15-marker block files are assembled into one continuous block of haplotypes for the entire chromosome. These extended haplotypes can then be used for further analysis.
  • haplotype assembly algorithms take the consensus estimate of the allele call at each marker over all separate estimations (most markers are estimated 15 different times as the 15 marker blocks pass over their position).
  • the haplotype frequencies among patients are compared to those among the controls using LDSTATS, a program that assesses the association of haplotypes with the disease.
  • Such program defines haplotypes using multi-marker windows that advance across the marker map in one-marker increments. Such windows can be 1 , 3, 5, 7 or 9 markers wide, and all these window sizes are tested concurrently. Larger multi-marker haplotype windows can also be used.
  • At each position the frequency of haplotypes in cases is compared to the frequency of haplotypes in controls.
  • Such allele frequency differences for single marker windows can be tested using Pearson's Chi-square with any degree of freedom.
  • Multi-allelic haplotype association can be tested using Smith's normalization of the square root of Pearson's Chi-square. Such significance of association can be reported in two ways:
  • P-values of association for each specific marker are calculated as a pooled P-value across all haplotype windows in which they occur.
  • the pooled P- value is calculated using an expected value and variance calculated using a permutation test that considers covariance between individual windows.
  • Such pooled P-values can yield narrower regions of gene location than the window data (see Example 3 herein for details on various analysis methods, such as LDSTATS v2.0 and v4.0).
  • conditional haplotype and subtype analyses can be performed on subsets of the original set of cases and controls using the program LDSTATS.
  • conditional analyses the selection of a subset of cases and their matched controls can be based on the carrier status of cases at a gene or locus of interest (see conditional analysis section in Example 3 herein).
  • conditional haplotypes can be derived, such as protective haplotypes and risk haplotypes.
  • Step 6 SNP and DNA polymorphism discovery
  • all the candidate genes and regions identified in step 5 are sequenced for polymorphism identification.
  • the entire region, including all introns, is sequenced to identify all polymorphisms.
  • the candidate genes are prioritized for sequencing, and only functional gene elements (promoters, conserved noncoding sequences, exons and splice sites) are sequenced.
  • previously identified polymorphisms in the candidate regions can also be used.
  • SNPs from dbSNP, or others can also be used rather than resequencing the candidate regions to identify polymorphisms.
  • the discovery of SNPs and DNA polymorphisms generally comprises a step consisting of determining the major haplotypes in the region to be sequenced.
  • the preferred samples are selected according to which haplotypes contribute to the association signal observed in the region to be sequenced.
  • the purpose is to select a set of samples that covers all the major haplotypes in the given region.
  • Each major haplotype is preferably analyzed in at least a few individuals.
  • Any analytical procedure may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention.
  • allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Any means of mutation detection or discrimination may be used. For instance, DNA sequencing, scanning methods, hybridization, extension based methods, incorporation based methods, restriction enzyme- based methods and ligation-based methods may be used in the methods of the invention.
  • Sequencing methods include, but are not limited to, direct sequencing, and sequencing by hybridization.
  • Scanning methods include, but are not limited to, protein truncation test (PTT), single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), cleavage, heteroduplex analysis, chemical mismatch cleavage (CMC), and enzymatic mismatch cleavage.
  • Hybridization-based methods of detection include, but are not limited to, solid phase hybridization such as dot blots, multiple allele specific diagnostic assay (MASDA), reverse dot blots, and oligonucleotide arrays (DNA Chips).
  • Solution phase hybridization amplification methods may also be used, such as Taqman.
  • Extension based methods include, but are not limited to, amplification refraction mutation systems (ARMS), amplification refractory mutation systems (ALEX), and competitive oligonucleotide priming systems (COPS).
  • Incorporation based methods include, but are not limited to, mini-sequencing and arrayed primer extension (APEX).
  • Restriction enzyme-based detection systems include, but are not limited to, restriction site generating PCR.
  • ligation based detection methods include, but are not limited to, oligonucleotide ligation assays (OLA).
  • Signal generation or detection systems that may be used in the methods of the invention include, but are not limited to, fluorescence methods such as fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence polarization as well as other chemiluminescence, electrochemiluminescence, Raman, radioactivity, colometric methods, hybridization protection assays and mass spectrometry methods.
  • Further amplification methods include, but are not limited to self sustained replication (SSR), nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and branched DNA (B-DNA).
  • SSR self sustained replication
  • NASBA nucleic acid sequence based amplification
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • B-DNA branched DNA
  • This step further maps the candidate regions and genes confirmed in the previous step to identify and validate the responsible polymorphisms associated with ENDOMETRIOSIS disease in the human population.
  • the discovered SNPs and polymorphisms of step 6 are ultrafine mapped at a higher density of markers than the GWS described herein using the same technology described in step 3.
  • Step 8 GeneMap construction
  • GeneMap for ENDOMETRIOSIS disease.
  • the gene content of this GeneMap is described in more detail below.
  • Such GeneMap can be used for other methods of the invention comprising the diagnostic methods described herein, the susceptibility to ENDOMETRIOSIS disease, the response to a particular drug, the efficacy of a particular drug, the screening methods described herein and the treatment methods described herein.
  • the GeneMap consists of genes and targets, in a variety of combinations, identified from the candidate regions listed in Table 1.
  • all genes from Tables 2-4 are present in the GeneMap.
  • the GeneMap consists of a selection of genes from Tables 2-4.
  • the genes of the invention (Tables 2-4) are arranged by candidate regions and by their chromosomal location. Such order is for the purpose of clarity and does not reflect any other criteria of selection in the association of the genes with ENDOMETRIOSIS.
  • the GeneMaps of the invention consists of a selection of genes from Tables 2-4 and a selection of genes that are interactors (direct or indirect) with the genes from the Tables.
  • the GeneMaps represent the knowledge that is needed for therapeutic and diagnostic intervention for a particular disease.
  • the GeneMaps aid in the selection of the best target to intervene in a disease state.
  • Each disease can be subdivided into various disease states and sub-phenotypes, thus various GeneMaps are needed to address various disease sub-phenopypes, and a clinical population can be stratified by sub-phenotype, which would be covered by a particular GeneMap.
  • genes identified in the WGAS and subsequent studies are evaluated using the Ingenuity Pathway Analysis application (IPA, Ingenuity systems) in order to identify direct biological interactions between these genes, and also to identify molecular regulators acting on those genes (indirect interactions) that could be also involved in ENDOMETRIOSIS.
  • IPA Ingenuity Pathway Analysis
  • the purpose of this effort is to decipher the molecules involved in contributing to ENDOMETRIOSIS.
  • nucleic acid sequences of the present invention may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, derivatives, mimetics or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns, genie regions, nongenic regions, and regulatory regions. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
  • nucleic acids described herein are used in certain embodiments of the methods of the present invention for production of RNA, proteins or polypeptides, through incorporation into cells, tissues, or organisms.
  • DNA containing all or part of the coding sequence for the genes described in Tables 2-4, or the SNP markers described in Tables 5-16, is incorporated into a vector for expression of the encoded polypeptide in suitable host cells.
  • the invention also comprises the use of the nucleotide sequence of the nucleic acids of this invention to identify DNA probes for the genes described in Tables 2-4 or the SNP markers described in Tables 5-16, PCR primers to amplify the genes described in Tables 2-4 or the SNP markers described in Tables 5-16, nucleotide polymorphisms in the genes described in Tables 2-4, and regulatory elements of the genes described in Tables 2-4.
  • nucleic acids of the present invention find use as primers and templates for the recombinant production of ENDOMETRIOSIS disease- associated peptides or polypeptides, for chromosome and gene mapping, to provide antisense sequences, for tissue distribution studies, to locate and obtain full length genes, to identify and obtain homologous sequences (wild-type and mutants), and in diagnostic applications.
  • an antisense nucleic acid or oligonucleotide is wholly or partially complementary to, and can hybridize with, a target nucleic acid (either DNA or RNA) having the sequence of SEQ ID NO:1 , NO:3 or any SEQ ID from any Tables of the invention.
  • a target nucleic acid either DNA or RNA
  • an antisense nucleic acid or oligonucleotide comprising 16 nucleotides can be sufficient to inhibit expression of at least one gene from Tables 2-4.
  • an antisense nucleic acid or oligonucleotide can be complementary to 5' or 3 1 untranslated regions, or can overlap the translation initiation codon (5 1 untranslated and translated regions) of at least one gene from Tables 2-4, or its functional equivalent.
  • the antisense nucleic acid is wholly or partially complementary to, and can hybridize with, a target nucleic acid that encodes a polypeptide from a gene described in Tables 2-4.
  • oligonucleotides can be constructed which will bind to duplex nucleic acid (i.e., DNA:DNA or DNA:RNA), to form a stable triple helix containing or triplex nucleic acid.
  • duplex nucleic acid i.e., DNA:DNA or DNA:RNA
  • triplex oligonucleotides can inhibit transcription and/or expression of a gene from Tables 2-4, or its functional equivalent (M. D. Frank-Kamenetskii et al., 1995).
  • Triplex oligonucleotides are constructed using the basepairing rules of triple helix formation and the nucleotide sequence of the genes described in Tables 2-4.
  • oligonucleotide refers to naturally- occurring species or synthetic species formed from naturally-occurring subunits or their close homologs.
  • the term may also refer to moieties that function similarly to oligonucleotides, but have non-naturally-occurring portions.
  • oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art.
  • At least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure that functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.
  • the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non- chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
  • Oligonucleotides may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some non- limiting examples of modifications at the 2' position of sugar moieties which are useful in the present invention include OH, SH, SCH3, F, OCH3, OCN, O(CH2), NH2 and O(CH2)n CH3, where n is from 1 to about 10.
  • oligonucleotides are functionally interchangeable with natural oligonucleotides or synthesized oligonucleotides, which have one or more differences from the natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with at least one gene from Tables 2-4 DNA or RNA to inhibit the function thereof.
  • the oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits.
  • a "subunit" is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds.
  • Antisense nucleic acids or oligonucleotides can be produced by standard techniques (see, e.g., Shewmaker et ai, U.S. Patent No. 6,107,065).
  • the oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Any other means for such synthesis may also be employed; however, the actual synthesis of the oligonucleotides is well within the abilities of the practitioner. It is also well known to prepare other oligonucleotides such as phosphorothioates and alkylated derivatives.
  • RNA e.g., mRNA
  • DNA oligonucleotide
  • oligonucleotide that hybridizes to mRNA from a gene described in Tables 2-4 can be used to target the mRNA for RnaseH digestion.
  • an oligonucleotide that can hybridize to the translation initiation site of the mRNA of a gene described in Tables 2-4 can be used to prevent translation of the mRNA.
  • oligonucleotides that bind to the double-stranded DNA of a gene from Tables 2-4 can be administered. Such oligonucleotides can form a triplex construct and inhibit the transcription of the DNA encoding polypeptides of the genes described in Tables 2-4. Triple helix pairing prevents the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described (see, e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY).
  • antisense oligonucleotides may be targeted to hybridize to the following regions: mRNA cap region; translation initiation site; translational termination site; transcription initiation site; transcription termination site; polyadenylation signal; 3' untranslated region; 5 1 untranslated region; 5' coding region; mid coding region; 3' coding region; DNA replication initiation and elondation sites.
  • the complementary oligonucleotide is designed to hybridize to the most unique 5' sequence of a gene described in Tables 2-4, including any of about 15-35 nucleotides spanning the 5' coding sequence.
  • the antisense oligonucleotide can be synthesized, formulated as a pharmaceutical composition, and administered to a subject.
  • the synthesis and utilization of antisense and triplex oligonucleotides have been previously described (e.g., Simon et al., 1999; Barre et al., 2000; Elez et al., 2000; Sauter et al., 2000).
  • expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population.
  • Transient expression may last for a month or more with a nonreplicating vector, and even longer if appropriate replication elements are included in the vector system.
  • Various assays may be used to test the ability of gene-specific antisense oligonucleotides to inhibit the expression of at least one gene from Tables 2-4.
  • mRNA levels of the genes described in Tables 2-4 can be assessed by Northern blot analysis (Sambrook et al., 1989; Ausubel et al., 1992; J. C. Alwine et al. 1977; I. M. Bird, 1998), quantitative or semi-quantitative RT-PCR analysis (see, e.g., W.M.
  • antisense oligonucleotides may be assessed by measuring levels of the polypeptide from the genes described in Tables 2-4, e.g., by western blot analysis, indirect immunofluorescence and immunoprecipitation techniques (see, e.g., J. M. Walker, 1998, Protein Protocols on cD-ROM, Humana Press, Totowa, NJ). Any other means for such detection may also be employed, and is well within the abilities of the practitioner.
  • mapping technologies include various methods which employ mapping technologies to map SNPs and polymorphisms.
  • this section comprises, but is not limited to, the description of mapping technologies that can be utilized to achieve the embodiments described herein.
  • Mapping technologies may be based on amplification methods, restriction enzyme cleavage methods, hybridization methods, sequencing methods, and cleavage methods using agents.
  • Amplification methods include: self sustained sequence replication (Guatelli et ai, 1990), transcriptional amplification system (Kwoh et al., 1989), Q- Beta Replicase (Lizardi et al., 1988), isothermal amplification (e.g. Dean et al., 2002; and Hafner et al., 2001), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of ordinary skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low number.
  • Restriction enzyme cleavage methods include: isolating sample and control DNA, amplification (optional), digestion with one or more restriction endonucleases, determination of fragment length sizes by gel electrophoresis and comparing samples and controls. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, e.g., U.S. Pat. No. 5,498,531 or DNAzyme e.g. U.S. Pat. No. 5,807,718, can be used to score for the presence of specific mutations by development or loss of a ribozyme or DNAzyme cleavage site.
  • Hybridization methods include any measurement of the hybridization or gene expression levels, of sample nucleic acids to probes corresponding to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, 100, 200, 500, 1000 or more genes, or ranges of these numbers, such as about 5-20, about 10-20, about 20- 50, about 50-100, or about 100-200 genes of Tables 2-4.
  • SNPs and SNP maps of the invention can be identified or generated by hybridizing sample nucleic acids, e.g., DNA or RNA, to high density arrays or bead arrays containing oligonucleotide probes corresponding to the polymorphisms of Tables 5-16 (see the Affymetrix arrays and lllumina bead sets at www.affymetrix.com and www.illumina.com and see Cronin et a/., 1996; or Kozal ef a/., 1996).
  • sample nucleic acids e.g., DNA or RNA
  • oligonucleotide analogue array can be synthesized on a single or on multiple solid substrates by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling (see Pirrung, U.S. Patent No. 5,143,854).
  • a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group.
  • a functional group e.g., a hydroxyl or amine group blocked by a photolabile protecting group.
  • Photolysis through a photolithogaphic mask is used selectively to expose functional groups which are then ready to react with incoming 5' photoprotected nucleoside phosphoramidites.
  • the phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group).
  • the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents.
  • High density nucleic acid arrays can also be fabricated by depositing pre-made or natural nucleic acids in predetermined positions. Synthesized or natural nucleic acids are deposited on specific locations of a substrate by light directed targeting and oligonucleotide directed targeting. Another embodiment uses a dispenser that moves from region to region to deposit nucleic acids in specific spots.
  • nucleic acid hybridization simply involves contacting a probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. See WO 99/32660. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA: DNA, RNA: RNA, or RNA: DNA) will form even where the annealed sequences are not perfectly complementary.
  • low stringency conditions e.g., low temperature and/or high salt
  • hybridization conditions may be selected to provide any degree of stringency.
  • hybridization is performed at low stringency to ensure hybridization and then subsequent washes are performed at higher stringency to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide.
  • Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level control, normalization control, mismatch controls, etc.).
  • the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity.
  • the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest.
  • Probes based on the sequences of the genes described above may be prepared by any commonly available method. Oligonucleotide probes for screening or assaying a tissue or cell sample are preferably of sufficient length to specifically hybridize only to appropriate, complementary genes or transcripts. Typically the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some cases, longer probes of at least 30, 40, or 50 nucleotides will be desirable.
  • oligonucleotide sequences that are complementary to one or more of the genes or gene fragments described in Tables 2-4 refer to oligonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequences of said genes. Such hybridizable oligonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes (see GeneChip ® Expression Analysis Manual, Affymetrix, Rev. 3, which is herein incorporated by reference in its entirety).
  • hybridizing specifically to or “specifically hybridizes” refers to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • a "probe” is defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • a variety of sequencing reactions known in the art can be used to directly sequence nucleic acids for the presence or the absence of one or more polymorphisms of Tables 5-16. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) or Sanger (1977). It is also contemplated that any of a variety of automated sequencing procedures can be utilized, including sequencing by mass spectrometry (see, e.g. PCT International Publication No. WO 94/16101 ; Cohen et al., 1996; and Griffin et a/., 1993), real-time pyrophosphate sequencing method (Ronaghi et a/., 1998; and Permutt et al., 2001) and sequencing by hybridization (see e.g. Drmanac et al., 2002).
  • mass spectrometry see, e.g. PCT International Publication No. WO 94/16101 ; Cohen et al., 1996; and Griffin et a/., 1993
  • RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes Other methods of detecting polymorphisms include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985).
  • the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing a wild-type sequence with potentially mutant RNA or DNA obtained from a sample.
  • the double- stranded duplexes are treated with an agent who cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of a mutation or SNP (see, for example, Cotton et al., 1988; and Saleeba et al., 1992).
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping polymorphisms.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches (Hsu et al., 1994).
  • Other examples include, but are not limited to, the MutHLS enzyme complex of E. coli (Smith and Modrich Proc. 1996) and CeI 1 from the celery (Kulinski et al., 2000) both cleave the DNA at various mismatches.
  • a probe based on a polymorphic site corresponding to a polymorphism of Tables 5-16 is hybridized to a cDNA or other DNA product from a test cell or cells.
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.
  • the screen can be performed in vivo following the insertion of the heteroduplexes in an appropriate vector. The whole procedure is known to those ordinary skilled in the art and is referred to as mismatch repair detection (see e.g. Fakhrai-Rad et al., 2004).
  • alterations in electrophoretic mobility can be used to identify polymorphisms in a sample.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of case and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence. The resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • RNA rather than DNA
  • the method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Kee et al., 1991).
  • the movement of mutant or wild-type fragments in a polyacrylamide gel containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., 1985).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum et al., 1987).
  • the mutant fragment is detected using denaturing HPLC (see e.g. Hoogendoom et al., 2000).
  • oligonucleotide primers may be prepared in which the polymorphism is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., 1986; Saiki et al., 1989).
  • Such oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • the amplification, the allele-specific hybridization and the detection can be done in a single assay following the principle of the 5' nuclease assay (e.g. see Livak et al., 1995).
  • the associated allele, a particular allele of a polymorphic locus, or the like is amplified by PCR in the presence of both allele-specific oligonucleotides, each specific for one or the other allele.
  • Each probe has a different fluorescent dye at the 5' end and a quencher at the 3' end.
  • the Taq polymerase via its 5' exonuclease activity will release the corresponding dyes. The latter will thus reveal the genotype of the amplified product.
  • Hybridization assays may also be carried out with a temperature gradient following the principle of dynamic allele-specific hybridization or like e.g. Jobs et al., (2003); and Bourgeois and Labuda, (2004).
  • the hybridization is done using one of the two allele-specific oligonucleotides labeled with a fluorescent dye, and an intercalating quencher under a gradually increasing temperature.
  • the probe is hybridized to both the mismatched and full-matched template.
  • the probe melts at a lower temperature when hybridized to the template with a mismatch.
  • the release of the probe is captured by an emission of the fluorescent dye, away from the quencher.
  • the probe melts at a higher temperature when hybridized to the template with no mismatch.
  • the temperature-dependent fluorescence signals therefore indicate the absence or presence of an associated allele, a particular allele of a polymorphic locus, or the like (e.g. Jobs et al., 2003).
  • the hybridization is done under a gradually decreasing temperature. In this case, both allele-specific oligonucleotides are hybridized to the template competitively. At high temperature none of the two probes are hybridized. Once the optimal temperature of the full-matched probe is reached, it hybridizes and leaves no target for the mismatched probe (e.g. Bourgeois and Labuda, 2004).
  • the allele-specific probes are differently labeled, then they are hybridized to a single PCR-amplified target. If the probes are labeled with the same dye, then the probe cocktail is hybridized twice to identical templates with only one labeled probe, different in the two cocktails, in the presence of the unlabeled competitive probe.
  • Oligonucleotides used as primers for specific amplification may carry the associated allele, a particular allele of a polymorphic locus, or the like, also referred to as "mutation" of interest in the center of the molecule, so that amplification depends on differential hybridization (Gibbs et al., 1989) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, 1993).
  • amplification may also be performed using Taq ligase for amplification (Barany, 1991). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known associated allele, a particular allele of a polymorphic locus, or the like at a specific site by looking for the presence or absence of amplification.
  • the products of such an oligonucleotide ligation assay can also be detected by means of gel electrophoresis.
  • the oligonucleotides may contain universal tags used in PCR amplification and zip code tags that are different for each allele.
  • the zip code tags are used to isolate a specific, labeled oligonucleotide that may contain a mobility modifier (e.g. Grossman et al., 1994).
  • allele-specific elongation followed by ligation will form a template for PCR amplification.
  • elongation will occur only if there is a perfect match at the 3' end of the allele-specific oligonucleotide using a DNA polymerase.
  • This reaction is performed directly on the genomic DNA and the extension/ligation products are amplified by PCR.
  • the oligonucleotides contain universal tags allowing amplification at a high multiplex level and a zip code for SNP identification.
  • the PCR tags are designed in such a way that the two alleles of a SNP are amplified by different forward primers, each having a different dye.
  • the zip code tags are the same for both alleles of a given SNPs and they are used for hybridization of the PCR- amplified products to oligonucleotides bound to a solid support, chip, bead array or like.
  • Fan et al. Cold Spring Harbor Symposia on Quantitative Biology, Vol. LXVIII, pp. 69-78 2003.
  • Another alternative includes the single-base extension/ligation assay using a molecular inversion probe, consisting of a single, long oligonucleotide (see e.g. Hardenbol et al., 2003).
  • the oligonucleotide hybridizes on both side of the SNP locus directly on the genomic DNA, leaving a one-base gap at the SNP locus.
  • the gap-filling, one-base extension/ligation is performed in four tubes, each having a different dNTP.
  • the oligonucleotide is circularized whereas unreactive, linear oligonucleotides are degraded using an exonuclease such as exonuclease I of E. coli.
  • the circular oligonucleotides are then linearized and the products are amplified and labeled using universal tags on the oligonucleotides.
  • the original oligonucleotide also contains a SNP-specific zip code allowing hybridization to oligonucleotides bound to a solid support, chip, and bead array or like. This reaction can be performed at a high multiplexed level.
  • the associated allele, a particular allele of a polymorphic locus, or the like is scored by single-base extension (see e.g. U.S. Pat. No. 5,888,819).
  • the template is first amplified by PCR.
  • the extension oligonucleotide is then hybridized next to the SNP locus and the extension reaction is performed using a thermostable polymerase such as ThermoSequenase (GE Healthcare) in the presence of labeled ddNTPs. This reaction can therefore be cycled several times.
  • the identity of the labeled ddNTP incorporated will reveal the genotype at the SNP locus.
  • the labeled products can be detected by means of gel electrophoresis, fluorescence polarization (e.g. Chen et al., 1999) or by hybridization to oligonucleotides bound to a solid support, chip, and bead array or like. In the latter case, the extension oligonucleotide will contain a SNP-specific zip code tag.
  • a SNP is scored by selective termination of extension.
  • the template is first amplified by PCR and the extension oligonucleotide hybridizes in the vicinity of the SNP locus, close to but not necessarily adjacent to it.
  • the extension reaction is carried out using a thermostable polymerase such as ThermoSequenase (GE Healthcare) in the presence of a mix of dNTPs and at least one ddNTP.
  • ThermoSequenase GE Healthcare
  • ThermoSequenase GE Healthcare
  • ThermoSequenase GE Healthcare
  • ThermoSequenase GE Healthcare
  • the extension product can then be detected by means of gel electrophoresis, in which case the extension products need to be labeled, or by mass spectrometry (see e.g. Storm et al., 2003).
  • SNPs are detected using an invasive cleavage assay (see U.S. Pat. No. 6,090,543).
  • oligonucleotides per SNP to interrogate but these are used in a two step-reaction. During the primary reaction, three of the designed oligonucleotides are first hybridized directly to the genomic DNA. One of them is locus-specific and hybridizes up to the SNP locus (the pairing of the 3' base at the SNP locus is not necessary).
  • the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a gene from Tables 2-4. Such methods may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention.
  • an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down- regulating expression of the nucleic acid in a cell.
  • Such cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium.
  • Some non-limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
  • RNA or mRNA is isolated by standard procedures such as those disclosed in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared as described above. Hybridization conditions are modified using known methods, such as those described by Sambrook et al., and Ausubel et al., as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip or a porous glass wafer.
  • the chip or wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize to the RNA.
  • agents which up or down regulate expression are identified.
  • the present invention provides methods for identifying agents that modulate at least one activity of the proteins described in Tables 2-4. Such methods may utilize any means of monitoring or detecting the desired activity.
  • an agent is said to modulate the expression of a protein of the invention if it is capable of up- or down- regulating expression of the protein in a cell.
  • Such cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium.
  • Some non-limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
  • the specific activity of a protein of the invention may be assayed in a cell population that has been exposed to the agent to be tested and compared to an unexposed control cell population.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and times.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with a probe, such as an antibody probe.
  • Antibodies and Antibody probes can be prepared by immunizing suitable mammalian (e.g. mice or transgenic mice) hosts utilizing appropriate immunization protocols using the proteins of the invention or antigen-containing fragments thereof. To enhance immunogenicity, these proteins or fragments can be conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL) may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • suitable adjuvants as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using standard methods, see e.g., Kohler & Milstein (1992) or modifications which affect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies can be screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies may be recovered from the culture supernatant or from the ascites supernatant.
  • Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant portion(s) can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive fragments, such as Fab or Fab' fragments, is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibody chains (light and heavy) can be cloned into the vector by methods known in the art.
  • the antibodies or fragments may also be produced, using current technology, by recombinant means.
  • Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras derived from multiple species.
  • Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras from multiple species, for instance, humanized antibodies.
  • the antibody can therefore be a humanized antibody or a human antibody, as described in U.S. Patent 5,585,089 or Riechmann et a/. (1988).
  • Phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for proteins, or fragments thereof, described in Tables 2-4. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al., EMBO J., 13:3245- 3260 (1994); Nissim et al., ibid, pp. 692-698 and by Griffiths et al., ibid, 12:725- 734.
  • the antibody of the invention also comprise humanized and human antibodies. Such antibodies are made by methods known in the art.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the protein of the invention alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a non- random basis which takes into account the sequence of the target site or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
  • the agents of the present invention can be, as examples, oligonucleotides, antisense polynucleotides, interfering RNA, peptides, peptide mimetics, antibodies, antibody fragments, small molecules, vitamin derivatives, as well as carbohydrates.
  • Peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene- encoded amino acids are to be included.
  • Another class of agents of the present invention includes antibodies or fragments thereof that bind to a protein encoded by a gene in Tables 2-4.
  • Antibody agents can be obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies (see section above of antibodies as probes for standard antibody preparation methodologies).
  • the present invention includes peptide mimetics that mimic the three-dimensional structure of the protein encoded by a gene from Tables 2-4.
  • Such peptide mimetics may have significant advantages over naturally occurring peptides, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half- life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity and others.
  • mimetics are peptide-containing molecules that mimic elements of protein secondary structure.
  • peptide mimetics The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are also referred to as peptide mimetics or peptidomimetics (Fauchere, 1986; Veber & Freidinger, 1985; Evans et a/., 1987) which are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • peptide mimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage using methods known in the art.
  • Labeling of peptide mimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptide mimetic that are predicted by quantitative structure-activity data and molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecule(s) to which the peptide mimetic binds to produce the therapeutic effect.
  • Derivitization (e.g., labeling) of peptide mimetics should not substantially interfere with the desired biological or pharmacological activity of the peptide mimetic.
  • the use of peptide mimetics can be enhanced through the use of combinatorial chemistry to create drug libraries.
  • the design of peptide mimetics can be aided by identifying amino acid mutations that increase or decrease binding of the protein to its binding partners. Approaches that can be used include the yeast two hybrid method (see Chien et al., 1991) and the phage display method.
  • the two hybrid method detects protein- protein interactions in yeast (Fields et al., 1989).
  • the phage display method detects the interaction between an immobilized protein and a protein that is expressed on the surface of phages such as lambda and M 13 (Amberg et al., 1993; Hogrefe et al., 1993). These methods allow positive and negative selection for protein-protein interactions and the identification of the sequences that determine these interactions.
  • the present invention also relates to methods for diagnosing ENDOMETRIOSIS or a related disease, preferably a subtype of ENDOMETRIOSIS, a predisposition to such a disease and/or disease progression.
  • the steps comprise contacting a target sample with (a) nucleic acid molecule(s) or fragments thereof and comparing the concentration of individual mRNA(s) with the concentration of the corresponding mRNA(s) from at least one healthy donor.
  • samples are, preferably, obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium.
  • Some non-limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
  • RNA is obtained from cells according to standard procedures and, preferably, reverse-transcribed.
  • a DNAse treatment in order to get rid of contaminating genomic DNA is performed.
  • the nucleic acid molecule or fragment is typically a nucleic acid probe for hybridization or a primer for PCR.
  • the person skilled in the art is in a position to design suitable nucleic acids probes based on the information provided in the Tables of the present invention.
  • the target cellular component i.e. mRNA, e.g., in brain tissue
  • Detection methods include Northern blot analysis, RNase protection, in situ methods, e.g.
  • PCR in situ hybridization
  • in vitro amplification methods PCR, LCR, QRNA replicase or RNA-transcription/amplification (TAS, 3SR), reverse dot blot disclosed in EP-B10237362
  • PCR in vitro amplification methods
  • TAS RNA-transcription/amplification
  • UAS reverse dot blot
  • products obtained by in vitro amplification can be detected according to established methods, e.g. by separating the products on agarose or polyacrylamide gels and by subsequent staining with ethidium bromide or any other dye or reagent.
  • the amplified products can be detected by using labeled primers for amplification or labeled dNTPs.
  • detection is based on a microarray.
  • the probes (or primers) can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.
  • the present invention also relates to the use of the nucleic acid molecules or fragments described above for the preparation of a diagnostic composition for the diagnosis of ENDOMETRIOSIS or a subtype or predisposition to such a disease.
  • the present invention also relates to the use of the nucleic acid molecules of the present invention for the isolation or development of a compound which is useful for therapy of ENDOMETRIOSIS.
  • the nucleic acid molecules of the invention and the data obtained using said nucleic acid molecules for diagnosis of ENDOMETRIOSIS might allow for the identification of further genes which are specifically dysregulated, and thus may be considered as potential targets for therapeutic interventions.
  • diagnostic might also be used for selection of patients that might respond positively or negatively to a potential target for therapeutic interventions (as for the pharmacogenomics and personalized medicine concept well know in the art; see prognostic assays text below).
  • the invention further provides prognostic assays that can be used to identify subjects having or at risk of developing ENDOMETRIOSIS.
  • a test sample is obtained from a subject and the amount and/or concentration of the nucleic acid described in Tables 2-4 is determined; wherein the presence of an associated allele, a particular allele of a polymorphic locus, or the likes in the nucleic acids sequences of this invention (see SEQ ID from Tables 5-16) can be diagnostic for a subject having or at risk of developing ENDOMETRIOSIS.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid, a cell sample, or tissue.
  • a biological fluid can be, but is not limited to saliva, serum, mucus, urine, stools, spermatozoids, vaginal secretions, lymph, amiotic liquid, pleural liquid and tears.
  • Cells can be, but are not limited to: ovarian cells, uterus cells and other cells of the reproductive system, hair cells, muscle cells, nervous cells, blood and vessels cells, dermis, epidermis and other skin cells, and various brain cells.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic acid such as antisense DNA or interfering RNA (RNAi), small molecule or other drug candidate) to treat ENDOMETRIOSIS.
  • an agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic acid such as antisense DNA or interfering RNA (RNAi), small molecule or other drug candidate
  • these assays can be used to predict whether an individual will have an efficacious response or will experience adverse events in response to such an agent.
  • such methods can be used to determine whether a subject can be effectively treated with an agent that modulates the expression and/or activity of a gene from Tables 2-4 or the nucleic acids described herein.
  • an association study may be performed to identify polymorphisms from Tables 5-16 that are associated with a given response to the agent, e.g., an efficacious response or the likelihood of one or more adverse events.
  • one embodiment of the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant expression or activity of a gene from Tables 2-4 in which a test sample is obtained and nucleic acids or polypeptides from Tables 2-4 are detected (e.g., wherein the presence of a particular level of expression of a gene from Tables 2-4 or a particular allelic variant of such gene, such as polymorphisms from Tables 5-16 is diagnostic for a subject that can be administered an agent to treat a disorder such as ENDOMETRIOSIS).
  • the method includes obtaining a sample from a subject suspected of having ENDOMETRIOSIS or an affected individual and exposing such sample to an agent.
  • the expression and/or activity of the nucleic acids and/or genes of the invention are monitored before and after treatment with such agent to assess the effect of such agent. After analysis of the expression values, one skilled in the art can determine whether such agent can effectively treat such subject.
  • the method includes obtaining a sample from a subject having or susceptible to developing ENDOMETRIOSIS and determining the allelic constitution of polymorphisms from Tables 5-16 that are associated with a particular response to an agent. After analysis of the allelic constitution of the individual at the associated polymorphisms, one skilled in the art can determine whether such agent can effectively treat such subject.
  • the methods of the invention can also be used to detect genetic alterations in a gene from Tables 2-4, thereby determining if a subject with the lesioned gene is at risk for a disease associated with ENDOMETRIOSIS.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration linked to or affecting the integrity of a gene from Tables 2-4 encoding a polypeptide or the misexpression of such gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of: (1) a deletion of one or more nucleotides from a gene from Tables 2-4; (2) an addition of one or more nucleotides to a gene from Tables 2-4; (3) a substitution of one or more nucleotides of a gene from Tables 2-4; (4) a chromosomal rearrangement of a gene from Tables 2-4; (5) an alteration in the level of a messenger RNA transcript of a gene from Tables 2-4; (6) aberrant modification of a gene from Tables 2-4, such as of the methylation pattern of the genomic DNA, (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a gene from Tables 2-4; (8) inappropriate post-translational modification of a polypeptide encoded by a gene from Tables 2-4; and (9) alternative promoter use.
  • a preferred biological sample is a peripheral blood sample obtained by conventional means from a subject.
  • Another preferred biological sample is a buccal swab.
  • Other biological samples can be, but are not limited to, urine, stools, vaginal secretions, lymph, amiotic liquid, pleural liquid and tears.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et a/., 1988; and Nakazawa et ai, 1994), the latter of which can be particularly useful for detecting point mutations in a gene from Tables 2-4 (see Abavaya et a/., 1995).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic DNA, mRNA, or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene from Tables 2-4 under conditions such that hybridization and amplification of the nucleic acid from Tables 2-4 (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic DNA, mRNA, or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with some of the techniques used for detecting a mutation, an associated allele, a particular allele of a polymorphic locus, or the like described in the above sections.
  • Other mutation detection and mapping methods are described in previous sections of the detailed description of the present invention.
  • the present invention also relates to further methods for diagnosing ENDOMETRIOSIS or a related disorder or subtype, a predisposition to such a disorder and/or disorder progression.
  • the steps comprise contacting a target sample with (a) nucleic molecule(s) or fragments thereof and determining the presence or absence of a particular allele of a polymorphism that confers a disorder-related phenotype (e.g., predisposition to such a disorder and/or disorder progression).
  • the presence of at least one allele from Tables 5- 16 that is associated with ENDOMETRIOSIS ("associated allele"), at least 5 or 10 associated alleles from Tables 5-16, at least 50 associated alleles from Tables 5- 16 at least 100 associated alleles from Tables 5-16, or at least 200 associated alleles from Tables 5-16 determined in the sample is an indication of ENDOMETRIOSIS disease or a related disorder, a disposition or predisposition to such kinds of disorders, or a prognosis for such disorder progression.
  • Such samples and cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium.
  • Some non- limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
  • alterations in a gene from Tables 2-4 can be identified by hybridizing sample and control nucleic acids, e.g., DNA or RNA, to high density arrays or bead arrays containing tens to thousands of oligonucleotide probes (Cronin et al., 1996; Kozal et al., 1996).
  • alterations in a gene from Tables 2-4 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al., (1996).
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations, associated alleles, particular alleles of a polymorphic locus, or the like. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants, mutations, alleles detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence a gene from Tables 2-4 and detect an associated allele, a particular allele of a polymorphic locus, or the like by comparing the sequence of the sample gene from Tables 2-4 with the corresponding wild-type (control) sequence (see text described in previous sections for various sequencing techniques and other methods of detecting an associated allele, a particular allele of a polymorphic locus, or the likes in a gene from Tables 2-4.
  • Such methods include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985) and alterations in electrophoretic mobility.
  • Examples of other techniques for detecting point mutations, an associated allele, a particular allele of a polymorphic locus, or the like include, but are not limited to, selective oligonucleotide hybridization, selective amplification, selective primer extension, selective ligation, single-base extension, selective termination of extension or invasive cleavage assay.
  • microsatellites can also be useful to detect the genetic predisposition of an individual to a given disorder.
  • Microsatellites consist of short sequence motifs of one or a few nucleotides repeated in tandem. The most common motifs are polynucleotide runs, dinucleotide repeats (particularly the CA repeats) and trinucleotide repeats. However, other types of repeats can also be used.
  • the microsatellites are very useful for genetic mapping because they are highly polymorphic in their length.
  • Microsatellite markers can be typed by various means, including but not limited to DNA fragment sizing, oligonucleotide ligation assay and mass spectrometry.
  • the locus of the microsatellite is amplified by PCR and the size of the PCR fragment will be directly correlated to the length of the microsatellite repeat.
  • the size of the PCR fragment can be detected by regular means of gel electrophoresis.
  • the fragment can be labeled internally during PCR or by using end-labeled oligonucleotides in the PCR reaction (e.g. Mansfield et a/., 1996).
  • the size of the PCR fragment is determined by mass spectrometry.
  • an oligonucleotide ligation assay can be performed.
  • the microsatellite locus is first amplified by PCR.
  • different oligonucleotides can be submitted to ligation at the center of the repeat with a set of oligonucleotides covering all the possible lengths of the marker at a given locus (Zirvi et ai, 1999).
  • Another example of design of an oligonucleotide assay comprises the ligation of three oligonucleotides; a 5' oligonucleotide hybridizing to the 5' flanking sequence, a repeat oligonucleotide of the length of the shortest allele of the marker hybridizing to the repeated region and a set of 3' oligonucleotides covering all the existing alleles hybridizing to the 3' flanking sequence and a portion of the repeated region for all the alleles longer than the shortest one.
  • the 3' oligonucleotide exclusively hybridizes to the 3' flanking sequence (U.S. Pat. No. 6,479,244).
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid selected from the SEQ ID of Tables 5-16, or antibody reagent described herein, which may be conveniently used, for example, in a clinical setting to diagnose patient exhibiting symptoms or a family history of a disorder or disorder involving abnormal activity of genes from Tables 2-4.
  • the present invention provides methods of treating a disease associated with ENDOMETRIOSIS disease by expressing in vivo the nucleic acids of at least one gene from Tables 2-4.
  • These nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below.
  • the nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell.
  • the nucleic acids encoding a gene from Tables 2-4, under the control of a promoter, then express the encoded protein, thereby mitigating the effects of absent, partial inactivation, or abnormal expression of a gene from Tables 2-4.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • RNA or DNA based viral systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non- dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of c/s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian lmmuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., 1992; Johann et al., 1992; Sommerfelt et al., 1990; Wilson et a/., 1989; Miller et al., 1999;and PCT/US94/05700).
  • adenoviral based systems are typically used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., 1987; U.S. Pat. No. 4,797,368; WO 93/24641 ; Kotin, 1994; Muzyczka, 1994).
  • rAAV Recombinant adeno-associated virus vectors
  • All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al., 1998, Kearns et al., 1996).
  • Ad vectors Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used in transient expression gene therapy; because they can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply the deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle tissues. Conventional Ad vectors have a large carrying capacity.
  • Ad vector An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., 1998). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., 1996; Sterman et al., 1998; Welsh et al., 1995; Alvarez et al., 1997; Topf et al., 1998.
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • the gene therapy vector be delivered with a high degree of specificity to a particular tissue type.
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Han et al., 1995 reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., Fab or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • antibody fragments e.g., Fab or Fv
  • nonviral vectors Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, and tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
  • a nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et a/., 1994; and the references cited therein for a discussion of how to isolate and culture cells from patients).
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba ef a/., 1992).
  • Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells).
  • T cells CD4+ and CD8+
  • CD45+ panB cells
  • GR-1 granulocytes
  • lad differentiated antigen presenting cells
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells, as described above.
  • the nucleic acids from Tables 2-4 are administered in any suitable manner, preferably with the pharmaceutically acceptable carriers described above. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route (see Samulski et a/., 1989).
  • the present invention is not limited to any method of administering such nucleic acids, but preferentially uses the methods described herein.
  • the present invention further provides other methods of treating ENDOMETRIOSIS disease such as administering to an individual having ENDOMETRIOSIS disease an effective amount of an agent that regulates the expression, activity or physical state of at least one gene from Tables 2-4.
  • An "effective amount" of an agent is an amount that modulates a level of expression or activity of a gene from Tables 2-4, in a cell in the individual at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more, compared to a level of the respective gene from Tables 2-4 in a cell in the individual in the absence of the compound.
  • the preventive or therapeutic agents of the present invention may be administered, either orally or parenterally, systemically or locally.
  • intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppositories, intestinal lavage, oral enteric coated tablets, and the like can be selected, and the method of administration may be chosen, as appropriate, depending on the age and the conditions of the patient.
  • the effective dosage is chosen from the range of 0.01 mg to 100 mg per kg of body weight per administration. Alternatively, the dosage in the range of 1 to 1000 mg, preferably 5 to 50 mg per patient may be chosen.
  • the therapeutic efficacy of the treatment may be monitored by observing various parts of the reproductive system and other body parts, or any other monitoring methods known in the art. Other ways of monitoring efficacy can be, but are not limited to monitoring pelvic pain, fertility, ovarian cysts formation and progression, or any other ENDOMETRIOSIS related symptom.
  • the present invention further provides a method of treating an individual clinically diagnosed with ENDOMETRIOSES' disease.
  • the methods generally comprises analyzing a biological sample that includes a cell, in some cases, a cell, from an individual clinically diagnosed with ENDOMETRIOSIS disease for the presence of modified levels of expression of at least 1 gene, at least 10 genes, at least 50 genes, at least 100 genes, or at least 200 genes from Tables 2-4.
  • a treatment plan that is most effective for individuals clinically diagnosed as having a condition associated with ENDOMETRIOSIS disease is then selected on the basis of the detected expression of such genes in a cell.
  • Treatment may include administering a composition that includes an agent that modulates the expression or activity of a protein from Tables 2-4 in the cell.
  • the invention further provides a method for predicting a patient's likelihood to respond to a drug treatment for a condition associated with ENDOMETRIOSIS disease, comprising determining whether modified levels of a gene from Tables 2-4 is present in a cell, wherein the presence of protein is predictive of the patient's likelihood to respond to a drug treatment for the condition.
  • Examples of the prevention or improvement of symptoms accompanied by ENDOMETRIOSIS disease that can monitored for effectiveness include prevention or improvement of pelvic pain, infertility, or any other ENDOMETRIOSIS related symptom.
  • the invention also provides a method of predicting a response to therapy in a subject having ENDOMETRIOSIS disease by determining the presence or absence in the subject of one or more markers associated with ENDOMETRIOSIS disease described in Tables 5-16, diagnosing the subject in which the one or more markers are present as having ENDOMETRIOSIS disease, and predicting a response to a therapy based on the diagnosis e.g., response to therapy may include an efficacious response and/or one or more adverse events.
  • the invention also provides a method of optimizing therapy in a subject having ENDOMETRIOSIS disease by determining the presence or absence in the subject of one or more markers associated with a clinical subtype of ENDOMETRIOSIS disease, diagnosing the subject in which the one or more markers are present as having a particular clinical subtype of ENDOMETRIOSIS disease, and treating the subject having a particular clinical subtype of ENDOMETRIOSIS disease based on the diagnosis.
  • treatment for the pelvic pain or infertility subtypes of ENDOMETRIOSIS is an example, treatment for the pelvic pain or infertility subtypes of ENDOMETRIOSIS.
  • Example 1 Identification of cases and controls
  • Reduced allelic heterogeneity will act to increase relative risk imparted by the remaining alleles and so increase the power of case/control studies to detect genes and gene alleles involved in complex disorders within the Quebec population.
  • the specific combination of age in generations, optimal number of founders and large present population size makes the QFP optimal for LD-based gene mapping.
  • the extraction method yielded high molecular weight DNA, and the quality of every DNA sample was verified by agarose gel electrophoresis. Genomic DNA appeared on the gel as a large band of very high molecular weight. The remaining two buffy coats were stored at -80 0 C as backups.
  • Genotyping was performed using the QLDM-Max SNP map using lllumina's Infinium-ll technology Single Sample Beadchips.
  • the QLDM-Max map contains 374,187 SNPs.
  • the SNPs are contained in the lllumina HumanHap-300 arrays plus two custom SNP sets of approximately 30,000 markers each.
  • the HumanHap-300 chip includes 317,503 tag SNPs derived from the Phase I HapMap data.
  • the additional (approx.) 60,000 SNPs were selected by to optimize the density of the marker map across the genome matching the LD pattern in the Quebec Founder Population, as established from previous studies at Genizon, and to fill gaps in the lllumina HumanHap-300 map.
  • the SNPs were genotyped on the 459 trios for a total of ⁇ 515,255,499 genotypes.
  • the genotyping information was entered into a Unified Genotype Database (a proprietary database under development) from which it was accessed using custom-built programs for export to the genetic analysis pipeline. Analyses of these genotypes were performed with the statistical tools described in Example 3. The GWS and the different analyses permitted the identification of candidate chromosomal regions linked to ENDOMETRIOSIS disease (Table 1).
  • Haplotypes will were estimated from the case/control genotype data using ggplem a modified version of the PL-EM algorithm.
  • the programs Qeno2patctr and tapper determined case and control genotypes and prepared the data in the input format for PL-EM.
  • An EM . algorithm module consisting of several applications was used to resolve phase ambiguities.
  • PLEMPre first recoded the genotypes for input into the PL-EM algorithm which used an 11- marker sliding block for haplotype estimation and deposited the constructed haplotypes into a file, happatctr which was the input file for haplotype association analysis performed by the program, LDSTATS.
  • the program GeneWriter was used to create a case-control genotype file, penopatctr, which was the input for the program, SiNGLETYPE, which was used to perform single marker case-control association analysis.
  • Haplotype association analysis was performed using the program LDSTATS.
  • LDSTATS tests for association of haplotypes with the disease phenotype.
  • the algorithms LDSTATS (v2.0) and LDSTATS (v4.0) define haplotypes using multi-marker windows that advance across the marker map in one-marker increments. Windows of size 1 , 3, 5, 7, and 9 were analyzed. At each position the frequency of haplotypes in cases and controls was determined and a chi-square statistic was calculated from case control frequency tables.
  • LDSTATS v2.0 the significance of the chi-square for single marker and 3-marker windows was calculated as Pearson's chi-square with degrees of freedom. Larger windows of multi-allelic haplotype association were tested using Smith's normalization of the square root of Pearson's Chi-square.
  • LDSTATS v4.0 calculates significance of chi-square values using a permutation test in which case-control status is randomly permuted until 350 permuted chi-square values are observed that are greater than or equal to chi- square value of the actual data. The P value is then calculated as 350 / the number of permutations required.
  • Tables 5.1-16.1 lists the results for association analysis using LDSTATs (v2.0 and v4.0) for the candidate regions described in Table 1 based on the genome wide scan genotype data for various subphenotypes from the QFP cases and controls. For each one of these regions, we report in Tables 5.2- 16.2 the allele frequencies and the relative risk (RR) for the haplotypes contributing to the best signal at each SNP in the region.
  • the program SINGLETYPE was used to calculate both allelic and genotype association for each single marker, one at a time using the genotype data in the file, genopatctr as input. Allelic association was tested using a 2 X 2 contingency table comparing allele 1 in cases and controls and allele 2 in cases and controls and genotype association was tested using a 2 X 3 contingency table comparing genotype 11 in cases and controls, genotype 12 in cases and controls and genotype 22 in cases and controls. SINGLETYPE was also used to test dominant and recessive models (11 and 12 genotypes combined vs. 22; or 22 and 12 genotypes combined vs. 11).
  • Conditional analyses were performed on subsets of the original set of 511 cases using the program LDSTATS (v2.0). The selection of a subset of cases and their matched controls was based on the carrier status of cases at a gene or locus of interest.
  • the set consisted of genotypes 1/2 and 2/2. Using this set, we partitioned the cases into two groups; the first group consisting of those cases that were carrier of a risk genotype and the second group consisting of the remaining cases, the non-carriers. The resulting sample sizes were respectively 329 and 181.
  • LDSTAT (v2.0) was run in each group and regions showing association with endometriosis are reported in Table 12.1. Regions associated with endometriosis in the group of carriers (has_PRKCE-1_cr) indicate the presence of an epistatic interaction between risk factors in those regions and risk factors in PRKCE (Table 12.2).
  • a third conditional analysis was performed using gene DNAH5 on chromosome 5.
  • the most significant association in DNAH5, using build 36, was obtained with a SNP corresponding to SEQ ID 5001 (see Table below for conversion to the specific DNA genotypes used).
  • LDSTAT (v2.0) was run in each group and a region showing association with endometriosis is reported in Table 14.1.
  • a region associated with endometriosis in the group of carriers (has_DNAH5-1_cr) indicates the presence of an epistatic interaction between risk factors in the region and risk factors in DNAH5 (Table 14.2).
  • a fourth conditional analysis was performed using gene SYNE1 on chromosome 6.
  • the most significant association signal in SYNE1 using build 36, was obtained with a SNP corresponding to SEQ ID 5106 (see Table below for conversion to the specific DNA alleles used).
  • LDSTAT (v2.0) was run in each group and regions showing association with endometriosis are reported in Table 15.1 for the group of carriers and in Table 16.1 for the group of non-carriers.
  • Regions associated with endometriosis in the group of carriers (has_SYNE1-1_cr) indicate the presence of an epistatic interaction between risk factors in the region and risk factors in SYNE1 (Table 15.2).
  • a region associated with endometriosis in the group of non-carriers (not_SYNE1-1_cr) indicates the existence of risk factors acting independently of SYNE1 (Table 16.2).
  • a unique consensus sequence was constructed for each splice variant and a trained reviewer assessed each alignment. This assessment included examination of all putative splice junctions for consensus splice donor/acceptor sequences, putative start codons, consensus Kozak sequences and upstream in-frame stops, and the location of polyadenylation signals. In addition, conserved noncoding sequences (CNSs) that could potentially be involved in regulatory functions were included as important information for each gene. The genomic reference and exon sequences were then archived for future reference. A master assembly that included all splice variants, exons and the genomic structure was used in subsequent analyses (i.e., analysis of polymorphisms). Table 3 lists gene clusters based on the publicly available EST and cDNA clustering algorithm, ECGene.
  • GeneMap contains networks highly relevant to ENDOMETRIOSIS as well as many genes under hormonal control.
  • the following examples of the emerging GeneMaps includes signaling pathways in cell proliferation, apoptosis, cell cycle, cell communication, cell structure, motility and hormonal regulation. Many of the identified regions contain genes involved in biologically relevant pathways, or associated conditions such as an oncogenesis-like mechanism, angiogenesis and infertility.
  • Angiogenesis might also play an important role in the pathogenesis of ENDOMETRIOSIS. It is viewed as a major prerequisite for the initiation and progression of the disease: known role in the survival of the implants and the development of ENDOMETRIOSIS. Anti- angiogenic agents may provide a novel therapeutic approach for the treatment of ENDOMETRIOSIS.
  • the genes from the observed GWAS results herein that may explain the Angiogenesis connection are RAF1 , PPARG, PRKCE, PRKD1 , PIK3C2A (Stage III/IV subphenotype) and SMOC2 (Not lnfertlity subphenotype).
  • ENDOMETRIOSIS is an estrogen-dependent disease and it is known that treatments tend to suppress estrogen synthesis.
  • Several of the identified pathways include genes that are regulated by or involved in the regulation of estrogen signaling: RAF1 , PRKCE, KCNQ3, AVPR2 (from conditional, epistatic to SYNE1) and ACE2 (from conditional, heterogeneity to SYNE1).
  • SYNE1 , KCNQ3, PRICKLE1 and SLC8A1 are GWAS genes involved in cell structure.
  • PPFIBP1 Stage III/IV subphenotype
  • PPFIBP1 is involved in focal adhesions, tumor invasiveness and metastasis.
  • Infertility Kinetics between endometrial/fallopian ciliated cells and uterine contractions may be important for normal function of fertilization and menstruation cycle.
  • Two of the genes, DNAH5 and DNAHL1 code for cilia motor proteins.
  • a study on ultrastructural aspects of endometrium in infertile women with septate uterus have shown irregular nonciliated cells with rare microvilli, incomplete ciliogenesis on ciliated cells, and decrease in the ciliated:nonciliated cell ratio.
  • Oncoqenesis-like mechanism Although ENDOMETRIOSIS is not a cancer, molecular and/or regulatory mechanisms responsible for the development of the disease may be similar. From the analyses of the GWAS data, it was found that the downstream genes that may explain the oncogenesis connection of ENDOMETRIOSIS include RAF1 , MCM3AP, MAD2L2 and H2AFY. RAF 1 is an oncogene while the other genes are respectively implicated in DNA replication, cell division and gene silencing, all activities that are known to be important in the development of tumorogenesis.
  • anti-angiogenic factors are used as an experimental treatment in animal models. They have been shown to cause regression and/or inhibition of the growth of endometriotic lesions. Most antiangiogenic agents have been discovered by identifying endogenous molecules that inhibit endothelial cells growth. This traditional approach has produced a number of anti-angiogenics; platelet factor-4 (PF4), thrombospondin, tumour necrosis factor (TNF)-a, interferon-c-inducible protein-10 (IP-10), angiostatin, endostatin, vasostatin, bactericidal-permeability increasing protein (BPI).
  • platelet factor-4 PF4
  • thrombospondin thrombospondin
  • TNF tumour necrosis factor
  • IP-10 interferon-c-inducible protein-10
  • BPI bactericidal-permeability increasing protein
  • Anti-angiogenic agents may provide a novel therapeutic approach for the treatment of ENDOMETRIOSIS.
  • PPARG is one of the genes identified on our GWAS study.
  • PPARG is a member of the peroxisome proliferator-activated receptor subfamily of nuclear receptors.
  • PPARG is a regulator of adipocyte differentiation and has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer.
  • Thiazolidinediones (TZD) are artificial ligands of PPARs, and are used clinically as anti-diabetic drugs. It has been shown that PPAR- ⁇ and -v are expressed by peritoneal macrophages isolated from ENDOMETRIOSIS patients. Also, the PPARG ligand rosiglitazone inhibit angiogenesis in tumors.
  • ENDOMETRIOSIS is an estrogen-dependent disease. Endometriotic implants are dependent on estrogen for their maintenance and growth. Treatments tend to suppress estrogen synthesis.
  • genes that are regulated by or involved in the regulation of estrogen signaling include PRKCE, RAF1 , KCNQ3 and AVPR2 (from conditional analysis herein).
  • Also numerous network genes in the described Genemaps are targeted by estrogen. These are IQGAP1 , SRC, EP300, SP1 , E2F1 , MAP2K1 , HEXIM1 , NTS, OXT and OXTR.
  • RAF1 Another compound that specifically targets RAF1 is XL281. Phase 1 trial in patients with advanced solid tumors is ongoing. Similar compound is XL147, which selectively targets PI3K. A Phase 1 trial in patients with solid tumors is ongoing. Also drugs that are PKC inhibitor, which inhibit PRKCE and / or PRKD1 (both genes identified in the GWA study), are used for various indications. MAPK inhibitors (MAP2K1 is a network gene) are also drugs tested for multiple tumors and advanced cancers.
  • EP300 Another network gene in the described Genemap is EP300, a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation.
  • EP300 is a target for histone deacetylase inhibitors, and is used in cancer therapy.
  • GWAS genome wide association study
  • the UniGene database contains information regarding the tissue source for ESTs and cDNAs contributing to individual clusters. This information was extracted and summarized to provide an indication in which tissues the gene was expressed. Particular emphasis was placed on annotating the tissue source for bona fide ESTs, since many ESTs mapped to Unigene clusters are artifactual.
  • SAGE and microarray data also curated at NCBI (Gene Expression Omnibus), provided information on expression profiles for individual genes. Particular emphasis was placed on identifying genes that were expressed in tissues known to be involved in the pathophysiology of endometriosis.
  • the first one was a RT-PCR based semiquantitative gene expression profiling method that could be applied to a large number of target sequences (genes, transcripts, ESTs) over a panel of 24 selected tissues.
  • the second was to map expression sites of mouse transcripts orthologous to a small set of human disease candidate genes in the mouse embryo (day 10.5, 12.5 and 15.5), in the postnatal stages (day 1 and 10) and at adulthood using in situ hybridization (ISH) method.
  • ISH in situ hybridization
  • Total human RNA samples from 24 different tissues Total RNA sample were purchased from commercial sources (Clontech, Stratagene) and used as templates for first-strand cDNA synthesis with the High-Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions.
  • a standard PCR protocol was used to amplify genes of interest from the original sample (50 ng cDNA); three serial dilutions of the cDNA samples corresponding to 5, 0.5 and 0.05 ng of cDNA were also tested. PCR products were separated by electrophoresis on a 96-well agarose gel containing ethidium bromide followed by UV imaging.
  • the serial dilutions of the cDNA provided semi-quantitative determination of relative mRNA abundance.
  • Tissue expression profiles were analyzed using standard gel imaging software (Alphalmager 2200); mRNA abundance was interpreted according to the presence of a PCR product in one or more of the cDNA sample dilutions used for amplification.
  • a PCR product present in all the cDNA dilutions i.e. from 50 to 0.05 ng cDNA
  • a PCR product only detectable in the original undiluted cDNA sample i.e., 50 ng cDNA
  • For each target gene one or more gene-specific primer pairs were designed to span at least one intron when possible. Multiple primer-pairs targeting the same gene allowed comparison of the tissue expression profiles and controlled for cases of poor amplification.
  • Tissue slices were mounted on glass microscope slides, fixed in formaldehyde and hybridized with 35 S-labeled cRNA probes. Antisense cRNA generated positive signals whereas sense cRNA (identical to mRNAs) generated negative (control) signals.
  • sense cRNA identical to mRNAs
  • control control signals.
  • cDNA clones of mouse orthologs to human genes H2afy, Mad2l2, Mcm3AP and Nrxni were obtained from commercial source (Open Biosystem).
  • DNA fragments to be used as templates for the cRNA probes synthesis were amplified by PCR and cloned into pGEM-7Zf(+)/LIC-F (ATCC #87048).
  • the templates for the antisense cRNA probes synthesis were generated by PCR using forward primers located at the 5' end of the cloned DNA fragments and a reverse primer located upstream of the SP6 polymerase promoter (in the vector).
  • the templates for the sense (control) cRNA probes synthesis were generated by PCR using a forward primer located upstream of the T7 promoter (in the vector) and reverse primers located at the 3' end of the cloned DNA fragments.
  • cRNA transcripts were synthesized in vitro from linear DNA fragments by run-off transcription with the SP6 or T7 RNA Polymerase from their respective promoters. Cold probe synthesis proved that DNA templates are functional and, hence, applied to radioactive probe synthesis labeled with 35 S- UTP (>1 ,000 Ci/mmol; Amersham).
  • Tissues were frozen-cut into 10- ⁇ m sections, mounted on gelatin- coated slides and stored at -80°C. Before ISH, they were fixed in 4% formaldehyde (freshly made from paraformaldehyde) in phosphate-buffered saline (PBS), treated with triethanolamine/acetic anhydride, washed and dehydrated with a series of ethanol. Hybridization and washing procedures.
  • PBS phosphate-buffered saline
  • Sections were hybridized overnight at 55°C in 50% deionized formamide, 0.3 M NaCI, 20 mM Tris-HCI, pH 7.4, 5 mM EDTA, 10 nM NaPO4, 10% dextran sulfate, 1 x Denhardt's, 50 ⁇ g/ml total yeast RNA, and 50-80,000 cpm/ ⁇ l 35 S-labeled cRNA probe.
  • the tissue was subjected to stringent washing at 65°C in 50% formamide, 2 x SSC, and 10 mM DTT, followed by washing in PBS before treatment with 20 ⁇ g/ml RNAse A at 37°C for 30 minutes.
  • the results are best viewed by darkfield illumination, with x2.5, x4, x10, x25 and 4Ox objectives; the silver grains can be localized over particular cells.
  • the antisense probe detects mRNA, and the sense control probe shows the background level of silver grains for the experiments.
  • H2afy displays a widespread if not ubiquitous distribution pattern in the midgestation stages e10.5, e12.5 and e15.5.
  • H2afy mRNA distribution pattern shows differentiation in high and low expression sites, to form a mosaic like pattern later at adulthood. More or less pronounced hybridization labeling occurs in the central nervous system, pituitary gland, adrenal gland, thymus, spleen, lymph nodes, testis, ovary and uterus.
  • the later, in pregnant female displays hybridization in the endometrium and decidua containing embryonic origin trophoblasts.
  • Complete picture of H2afy mRNA distribution in the adult mouse is shown in Table 19.
  • H2afy belongs to a class of ubiquitously expressed genes in the embryonic mouse which over a postnatal developmental differentiation process, acquire a cell and tissue specific pattern of distribution. This expression profil suggest that H2afy may play a role in both developmental and adulthood functions, including nervous, endocrine, immune and reproductive functions. Table 19: Detection of H2AFY mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
  • the adult brain and spinal cord synthesize much less Mad2l2 mRNA. Similar process has been observed in the peripheral nervous system sensory ganglia (dorsal root ganglia), ortosympathetic ganglia (paravertebral ganglia), olfactory neuroepithelium, retina in the eye and the organ of Corti in the ear, all displaying strong hybridization signal in p1 mice but weak in the adulthood. [000344] In contrast to the developmental mice, in the adulthood, low Mad2l2 expression levels were evident in most tissues, as shown in Table 21. Highest Mad2l2 mRNA levels were detected in the male testis seminiferous tubules.
  • the ovary and uterus contained low concentrations Mad2l2 mRNA.
  • pregnant mouse uterine tissue examined on day 5.5 and 7.5 post- coitum displayed increased levels of Mad2l2 mRNA around the sites of the conceptus implantation, where a decidua is formed. This observation suggests an induction of Mad2l2 expression specifically involved in the implantation or post- implantation processes.
  • the liver, spleen and kidney displayed low Mad2l2 levels.
  • Table 21 Detection of MAD2L2 mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
  • Table 22 Relative Correlation Between MAD2L2 and PCNA mRNA Ontogeny Distribution Patterns. Scale as in Table 21.
  • Mcm3ap gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 5 days and 17 days, respectively. Results are presented in Tables 23 and 24 and Figures T to W.
  • ISH results provide evidence for a Mcm3ap expression at low-level in the embryonic stages studied ( Figure T).
  • ISH results were readable following 5-day exposure of X-Ray Films, which is a limit of mRNA detection by a technique.
  • Mcm3ap displays a widespread if not ubiquitous distribution pattern observed from the midgestation stages e10.5, e12.5 and e15.5 to the adulthood with no significant changes in the pattern of tissue specificity and mRNA concentration.
  • slightly elevated hybridization levels were observed in some tissues such as the thymus and brain regions such as cerebellum and hippocampus of the newborn, postnatal and adult mice, these tissues are characterized by locally high density of cells.
  • mcm3ap concentration in these structures reflected rather the increasing cell density than gene expression regulation mechanism.
  • Table 24 The overview of mcm3ap mRNA distribution pattern in the adult mouse is shown in Table 24.
  • Mcm3ap belongs to a class of low-level ubiquitously expressed genes that maintain their mRNA distribution pattern and concentration level spanning over a life, in the mouse from the embryonic stages to the adulthood. Mcm3ap represents likely a housekeeping class of the genes.
  • Nrxni gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 2 days and 10 days, respectively. Results are presented in Tables 25 and 26 and Figures X to CC.
  • ISH results provide evidence for a Nrxni expression generally at high-level in the embryonic, newborn postnatal and adult mouse stages Table 25 and Figure X. Not detectable on day 10.5, ISH signal was evident on day 12.5 in the rudimental central (CNS) and peripheral (PNS) nervous system, persisting elevated along further developmental stages. The highest level of expression was noted to occur in CNS and PNS on postnatal day 10, followed by decline in the adult stage. Nrxni distribution in the adult stage is summarized in the Table 26. Briefly, presence of Nrxn1 mRNA was confined to neuronal cells of the grey matter. There was not detectable Nrxni mRNA in the white matter cells with oligodendrocyte topography.
  • Nrxni mRNA labeling the majority, but not all neuronal cells displayed Nrxni mRNA labeling. For example, there was not labeling in the Purkinje cells of the cerebellar folia and few other discreet regions but not shown here. For the above reason, Nrxni distribution cannot be defined as pan-neuronal, but rather neuron-specific.
  • Nrxni mRNA-labeled neurons in PNS ganglia To scrutinize Nrxni mRNA-labeled neurons in PNS ganglia the newborn and postnatal stages (p1 and p10) appeared especially useful when compared to adult stage: (1) there were higher gene expression levels evident in pp1 and p10 ganglia and (2) higher choice of sections that passed throughout relevant regions in comparison to low choice in the adult stage sections selection.
  • a list of Nrxni-labeled PNS ganglia include the sensory cranial ganglia such as the trigeminal ganglion as well as dorsal root ganglia. The ganglia of the sympathetic nervous system and visceral microganglia contributing to the plexus Auerbach expressed Nrxni mRNA.
  • the endocrine glands such as the pituitary gland and adrenal medulla displayed a low to medium Nrxni mRNA concentrations.
  • Nrxni belongs to a class of high-level neuronal- specific genes with distribution pattern following most CNS and PNS regions and two endocrine glands. In CNS and PNS, Nrxni occurs at concentrations that are up-regulated postnatally to a maximal levels measured on day 10. Nrxni represents likely a good neuronal marker, especially to the plexus Auerbach in the gut.

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Abstract

The present invention relates to the selection of a set of polymorphism markers for use in genome wide association studies based on linkage disequilibrium mapping. In particular, the invention relates to the fields of pharmacogenomics, diagnostics, patient therapy and the use of genetic haplotype information to predict an individual's susceptibility to ENDOMETRIOSIS disease and/or their response to a particular drug or drugs.

Description

GENEMAP OF THE HUMAN GENES ASSOCIATED WITH
ENDOMETRIOSIS
INVENTORS: Abdelmajid Belouchi, John Verner Raelson, Bruno Paquin, Sandie Briand, Daniel Dubois, Paul Van Eerdewegh, Jonathan Segal, Randall David Little and Tim Keith.
PRIORITY
[0001] This application is entitled to priority to U.S. Provisional Application No. 60/899,615, filed February 6, 2007 and U.S. Provisional Application No. 60/948,565, filed July 9, 2007, which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of genomics and genetics, including genome analysis and the study of DNA variations. In particular, the invention relates to the fields of pharmacogenomics, diagnostics, patient therapy and the use of genetic haplotype information to predict an individual's susceptibility to ENDOMETRIOSIS disease and/or their response to a particular drug or drugs, so that drugs tailored to genetic differences of population groups may be developed and/or administered to the appropriate population.
[0003] The invention also relates to a GeneMap for ENDOMETRIOSIS disease, which links variations in DNA (including both genie and non-genic regions) to an individual's susceptibility to ENDOMETRIOSIS disease and/or response to a particular drug or drugs. The invention further relates to the genes disclosed in the GeneMap (see Tables 2-4 and examples of the GeneMap in the Example section herein), which is related to methods and reagents for detection of an individual's increased or decreased risk for ENDOMETRIOSIS disease and related sub-phenotypes, by identifying at least one polymorphism in one or a combination of the genes from the GeneMap. Also related are the candidate regions identified in Table 1 , which are associated with ENDOMETRIOSIS disease. In addition, the invention further relates to nucleotide sequences of those genes including genomic DNA sequences, DNA sequences, single nucleotide polymorphisms (SNPs), other types of polymorphisms (insertions, deletions, microsatellites), alleles and haplotypes (see Sequence Listing and Tables 5-16).
[0004] The invention further relates to isolated nucleic acids comprising these nucleotide sequences and isolated polypeptides or peptides encoded thereby. Also related are expression vectors and host cells comprising the disclosed nucleic acids or fragments thereof, as well as antibodies that bind to the encoded polypeptides or peptides.
[0005] The present invention further relates to ligands that modulate the activity of the disclosed genes or gene products. In addition, the invention relates to diagnostics and therapeutics for ENDOMETRIOSIS disease, utilizing the disclosed nucleic acids, polymorphisms, chromosomal regions, GeneMaps, polypeptides or peptides, antibodies and/or ligands and small molecules that activate or repress relevant signaling events.
BACKGROUND OF THE INVENTION
[0006] ENDOMETRIOSIS is defined as the presence of endometrial-like tissue growing outside the uterine cavity. It is also associated with significant impairment in quality of life for affected women due to severe pain during menstruation and sexual intercourse, and infertility. The pathophysiology of ENDOMETRIOSIS remains enigmatic. As a result, current therapeutic strategies are mainly palliative and non-curative. Surgery is the first-line treatment to remove ovarian endometriomas and to correct ENDOMETRIOSIS-associated adhesions that can distort pelvic anatomy. Nevertheless, patients who undergo surgical procedures have recurrence of ENDOMETRIOSIS in up to 47% of cases and recurrence of adhesions in up to 89% of cases. New research treatments include the use of aromatase inhibitors together with progestin or together with oral contraceptives. However, ENDOMETRIOSIS recurs once all these treatments are stopped.
[0007] Medical pharmacological treatments such as the androgenic therapies, danazol and gestrinone, the constellation of GnRH agonists, buserelin, goserelin, leuprolide, nafarelin and triptorelin, GnRH antagonists, cetrorelix and abarelix, as well as the progestogens, including medroxyprogesterone acetate, induce lesion atrophy by suppressing the production of estrogen. These approaches are not without unwanted side effects. Danazol and gestrinone include weight gain, hirsuitism, acne, mood changes and metabolic effects on the cardiovascular system. The group of GnRH agonists and antagonists are found to cause a profound suppression of estrogen leading to vasomotor effects (hot flashes) and depletion of bone mineral density, which restricts their use to only six months of therapy. The group of progestogens, including medroxyprogesterone acetate, suppress the gonadotropins, but do not down-regulate ovarian estrogen production to the same extent as the GnRH analogues. The side effects include irregular bleeding, bloating, weight gain and metabolic effects on the cardiovascular system.
[0008] Current treatments do not address the root cause of the disease. Despite a preponderance of evidence showing inheritance of a risk for ENDOMETRIOSIS disease through epidemiological studies and genome wide linkage analyses, the genes affecting ENDOMETRIOSIS disease have yet to be discovered. There is a need in the art for identifying specific genes related to ENDOMETRIOSIS disease to enable the development of therapeutics that address the causes of the disease rather than relieving its symptoms. The failure in past studies to identify causative genes in complex diseases, such as ENDOMETRIOSIS disease, has been due to the lack of appropriate methods to detect a sufficient number of variations in genomic DNA samples (markers), the insufficient quantity of necessary markers available, and the number of needed individuals to enable such a study. The present invention addresses these issues. [0009] The present invention relates specifically to a set of ENDOMETRIOSIS disease-causing genes (GeneMap) and targets which present attractive points of therapeutic intervention and diagnostics.
[00010] In view of the foregoing, identifying susceptibility genes associated with ENDOMETRIOSIS disease and their respective biochemical pathways will facilitate the identification of diagnostic markers as well as novel targets for improved therapeutics. It will also improve the quality of life for those afflicted by this disease and will reduce the economic costs of these afflictions at the individual and societal level. The identification of those genetic markers would provide the basis for novel genetic tests and eliminate or reduce the therapeutic methods currently used. The identification of those genetic markers will also provide the development of effective therapeutic intervention for the battery of laboratory, phsychological and clinical evaluations typically required to diagnose ENDOMETRIOSIS. The present invention satisfies this need.
[00011] DESCRIPTION OF THE FILES CONTAINED ON THE CD-R
[00012] The contents of the submission on compact discs submitted herewith are incorporated herein by reference in their entirety: A compact disc copy of the Sequence Listing (COPY 1) (filename: GENI 024 01WO SeqList.txt, date recorded: February 06, 2008, file size: 14,920 kilobytes); a duplicate compact disc copy of the Sequence Listing (COPY 2) (filename: GENI 024 01WO SeqList.txt, date recorded: February 06, 2008, file size: 14,920 kilobytes); a duplicate compact disc copy of the Sequence Listing (COPY 3) (filename: GENI 024 01WO SeqList.txt, date recorded: February 06, 2008, file size: 14,920 kilobytes); a computer readable format copy of the Sequence Listing (CRF COPY) (filename: GENI 024 01WO SeqList.txt, date recorded: February 06, 2008, file size: 14,920 kilobytes).
[00013] Three compact disc copies (COPY 1 , COPY 2 and COPY3) of Tables 1-18 are herewith submitted and are incorporated herein by reference in their entirety. Each Table may be viewed properly in the Courier font. Each compact disc contains a copy of the following files:
[00014] filename: Table1.txt, date recorded: February 6, 2008, file size: 11 kilobytes;
[00015] filename: Table2.txt, date recorded: February 6, 2008, file size: 35 kilobytes;
[00016] filename: Table3.txt, date recorded: February 6, 2008, file size: 278 kilobytes;
[00017] filename: Table4.txt, date recorded: February 6, 2008, file size: 2 kilobytes;
[00018] filename: Table5.1.txt, date recorded: February 6, 2008, file size: 81 kilobytes;
[00019] filename: Table5.2.txt, date recorded: February 6, 2008, file size: 65 kilobytes;
[00020] filename: Table6.1.txt, date recorded: February 6, 2008, file size: 6 kilobytes;
[00021] filename: Table6.2.txt, date recorded: February 6, 2008, file size: 12 kilobytes;
[00022] filename: Table7.1.txt, date recorded: February 6, 2008, file size: 15 kilobytes;
[00023] filename: Table7.2.txt, date recorded: February 6, 2008, file size: 14 kilobytes;
[00024] filename: Table8.1.txt, date recorded: February 6, 2008, file size: 23 kilobytes; [00025] filename: Table8.2.txt, date recorded: February 6, 2008, file size: 22 kilobytes;
[00026] filename: Table9.1 , date recorded: February 6, 2008, file size: 4 kilobytes;
[00027] filename: Table9.2, date recorded: February 6, 2008, file size: 3 kilobytes;
[00028] filename: Table10.1 , date recorded: February 6, 2008, file size: 44 kilobytes;
[00029] filename: Table10.2, date recorded: February 6, 2008, file size: 17 kilobytes;
[00030] filename: TableH .1 , date recorded: February 6, 2008, file size: 31 kilobytes;
[00031] filename: Table11.2, date recorded: February 6, 2008, file size: 42 kilobytes;
[00032] filename: Table12.1 , date recorded: February 6, 2008, file size: 17 kilobytes;
[00033] filename: Table12.2, date recorded: February 6, 2008, file size: 11 kilobytes;
[00034] filename: Table13.1 , date recorded: February 6, 2008, file size: 27 kilobytes;
[00035] filename: Table13.2, date recorded: February 6, 2008, file size: 16 kilobytes;
[00036] filename: Table14.1 , date recorded: February 6, 2008, file size: 6 kilobytes; [00037] filename: Table14.2, date recorded: February 6, 2008, file size: 4 kilobytes;
[00038] filename: Table15.1 , date recorded: February 6, 2008, file size: 13 kilobytes;
[00039] filename: Table15.2, date recorded: February 6, 2008, file size: 8 kilobytes;
[00040] filename: Table16.1 , date recorded: February 6, 2008, file size: 4 kilobytes;
[00041] filename: Table16.2, date recorded: February 6, 2008, file size: 3 kilobytes;
[00042] filename: Table17, date recorded: February 6, 2008, file size: 16 kilobytes; and
[00043] filename: Table18, date recorded: February 6, 2008, file size: 11 kilobytes.
[00044] Brief Description of Drawings
[00045] Figure 1. Emerging endometriosis GeneMap.
[00046] Figure 2. Emerging endometriosis GeneMap with sub-phenotype.
[00047] Figure 3. Targeting the signaling pathway.
[00048] Figure 4. Mouse mRNA localization matrix applied to single and multiple mRNA localization assessment & comparative studies, cresyl violet staining. Figs. 4A-G: All-Stage, Whole-Body Sections throughout the embryonic (1 and 2), postnatal developmental stages (3 and 5) and adulthood (6 and 7). Fig. 4H: Adult Mouse Reproductive Organs: 1. Uterus, control; 2. Uterus, gestation day 5.5; 3. Uterus, gestation day 7.5; 4. Ovary; 5. Mammary gland; 6. Prostate; 7. Epididymis; 8. Testis; 9. Seminal vesicle; Fig. 41: Adult Mouse Tissue Array, General: 10. Brain, sagittal sections; 11. Thyroid; 12. Pituitary gland; 13. Adrenal gland; 14. Trigeminal ganglion; 15. Ovary; 16. Uterus; 17. Kidney; 18. Testis; 19. Thymus; 20. Seminal vesicle; 21. Salivary gland; 22. Urinary Bladder; 23. Lung; 24. Prostate; 25. Liver; 26. Gallbladder; 27. Epididymis; 28. Adipose tissue; Fig. 4J: Adult Mouse Brain Arrays.
[00049] Figure 5. H2AFY expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice. Figs. A-D) X-ray film autoradiography following hybridization with antisense riboprobe (Seq ID: 6717) after 3-day exposure, showing a pattern of H2AFY mRNA distribution seen as bright labeling on dark field. Fig. E) Control (sense, Seq ID: 6716) hybridization of the section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; E - eye; K - kidney; Li - liver; Lu - lung; OC - olfactory cavity; Sk - skin; St - stomach; Te - testis; Th - thymus; Ve - vertebrae; (s) - sense. Magnification x 1.6.
[00050] Figure 6. H2AFY expression in the adult mouse. Fig. 6A) Anatomical view of the adult mouse after staining with cresyl violet. Fig. 6B) X-ray film autoradiography after hybridization with antisense (Seq ID: 6717) riboprobe showing the presence of H2AFY mRNA in the brain, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination. Fig. 6C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Abbreviations: BM - bone marrow; Br - brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; LN - lymph node; Lu - lung; Ri - ribs; SG - salivary gland; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense. Magnification x 2.7.
[00051] Figure 7. H2AFY expression in the adult mouse tissue arrays. Fig. 7 A) X-ray film autoradiography after hybridization with antisense (Seq ID: 6717) riboprobe showing H2AFY mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Expression sites are evident in the ovary, control uterus and uterine tissue at gestation stages day 5.5 and 7.5. Fig. 7 B) H2AFY mRNA shown in the general tissue array (TA). Low to medium levels of expression are evident in most tissue including the brain, pituitary gland, adrenal gland, thyroid, testis, splee, kidney and prostate. High H2AFY mRNA concentrations occur in the thymus and ovary. Fig. 7 C) H2AFY mRNA in the brain tissue arrays; expression is evident in the olfactory lobe, hippocampus, hypothalamus and cerebellum. Fig. 7 D) Control (sense, Seq ID: 6716) hybridization of the section comparable to B. Abbreviations: Adr - adrenal gland; Br - brain; Hip - hippocampus; Cb - cerebellum; Hy - hypothalamus; K - kidney; Li - liver; Lu - lung; OL - olfactory lobe; Ov - ovary; Pit - pituitary gland; Pr - prostate; SG - salivary gland; Sp - spleen; Td - thyroid gland; Te - testis; Th - thymus; UB - urinary bladder; Ut - uterus; UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense. Magnification x 1.6.
[00052] Figure 8. H2AFY expression in the adult mouse brain hippocampus and cerebellum. Fig. 8A) Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the hippocampus seen as bright on darkfield illumination. Fig. 8B) Fragment of the hippocampus with H2AFY mRNA labeled area CA1 neurons (arrow) Fig. 8C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Fig. 8D) Cerebellum at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in Purkinje cells (arrows). Fig. 8E) Fragment of the cerebellum showing Purkinje cells layer at higher magnification (arrows). Fig. 8F) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to E. Abbreviations: CA1 - cornu Ammonis area 1 ; Cb - cerebellum; DG - dentate gyrus; PC - Purkinje cells layer; (s) - sense. Magnifications: (A and D) x 23; (B, C, E and F) x 405.
[00053] Figure 9. H2AFY expression in the adult mouse brain bone marrow and dorsal root ganglion. Fig. 9A) Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the bone marrow region and dorsal root ganglion seen as bright on darkfield illumination. Fig. 9B) Fragment of the bone marrow with H2FY mRNA labeled cells (arrow), bone unlabeled, at higher magnification. Fig. 9C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Fig. 9D) Dorsal root ganglion revealing the sensory neurons labeled (heavy arrows) at higher magnification, note the satellite glial cells unlabeled (small arrows). Fig. 9E) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to D. Abbreviations: B - bone; BM - bone marrow; DRG - dorsal root ganglion; Ve - vertebrae; (s) - sense. Magnifications: (A) x 50; (B to E) x 405.
[00054] Figure 10. H2AFY expression in the thymus. Fig. 10A) Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA as bright labeling under darkfield illumination. Fig. 10B) At higher magnification, it is seen that H2AFY mRNA labeling follow the cell density which is higher in the cortex and lower in the medulla. Fig. 10C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to A. Abbreviations: Cx - cortex; Me - medulla; (s) - sense. Magnifications: (A) x 25; (B and C) x 405.
[00055] Figure 11. H2AFY expression in the ovary. Fig. 11A) Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA seen as bright labeling under darkfield illumination mostly in the corpus luteum. Fig. 11 B) At higher magnification H2AFY mRNA labeling is seen in the corpus luteum cells and in the follicular cells (arrows). Theca cells seem to not express H2AFY. Fig. 11C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Abbreviations: CL - corpus luteum; FC - follicular cells; T - theca; (s) - sense. Magnifications: (A) x 25; (B and C) x 405.
[00056] Figure 12. H2AFY expression in the intact adult mouse uterus. Fig. 12A) Emulsion autoradiography at low magnification, after hybridization with antisense (Seq ID: 6717) riboprobe, showing H2AFY mRNA labeling in the endometrium epithelial cells layer (arrow) seen as bright under darkfield illumination. Fig. 12B) The same section seen at lightfield illumination and cresyl violet staining. Fig. 12C) Fragment of the uterine epithelium, labeled (arrow) at high magnification. Fig. 12D) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to A at darkfield illumination. Fig. 12E) The same section seen at lightfield illumination and cresyl violet staining. Fig. 12F) Fragment of the uterine epithelium following control (sense, Seq ID: 6716) hybridization. Abbreviations: E - endometrium; Ep - epithelium; M - myometrium, (as) - antisense; (s) - sense. Magnifications: (A, B, D and E) x 25; (C and F) x 614.
[00057] Figure 13. H2AFY expression in the female uterus 7,5 days pregnant. Fig. 13A) Emulsion autoradiography, after hybridization with antisense (Seq ID: 6717) riboprobe, throughout the peripheral region of the uterus. H2AFY mRNA labeling is present in mostly endometrium cells and much less in the myometrium. Fig. 13B) Centrally located deciduas with labeled giant cells originated from the ectoplacental cone (heavy arrows) and the presumptive trophoblasts of trophectoderm origin (small arrows). Fig. 13C) Control (sense, SEQIDPROBE2]) hybridization of an adjacent section comparable to B. Abbreviations: BV - blood vessels; E - endometrium; - H - hondrion; M - myometrium; (s) - sense. Magnification: x 380.
[00058] Figure 14. H2AFY expression in the testis. Fig. 14A) Emulsion autoradiography, after hybridization with antisense (Seq ID: 6717) riboprobe, throughout the testis showing H2AFY mRNA labeling as bright under lightfield illumination. Labeling is present in a proportion of seminiferous tubules (arrow). Fig. 14B) Fragment of the seminiferous tubule showing H2AFY mRNA labeling concentrated mostly in the layer of spermatogonia and much less in spermatocyte layer. There is no detectable labeling in the spermatozoa. Fig. 14C) Control (sense, Seq ID: 6716) hybridization of an adjacent section comparable to B. Abbreviations: Sc - spermatocyte; SfT - seminiferous tubule; Sg - spermatogonia; Sz - spermatozoa; (s) - sense. Magnifications: (A) x 25; (B and C) x 380.
[00059] Figure 15. MAD2L2 expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice. Figs. 15A-D) X-ray film autoradiography following hybridization with antisense (Seq ID: 6719) riboprobe after 4-day exposure, showing a pattern of MAD2L2 mRNA distribution seen as bright labeling on dark field. Fig. 15E: Control (sense, Seq ID: 6718) hybridization of the section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; DRG - dorsal root ganglia; K - kidney; Ov - ovary; Re - retina; SC - spinal cord; St - stomach; (s) - sense. Magnification x 1.6.
[00060] Figure 16. MAD2L2 expression in the adult mouse. Fig.16A) Anatomical view of the adult mouse after staining with cresyl violet. Fig.16B) X-ray film autoradiography following hybridization with antisense (Seq ID: 6719) riboprobe showing the presence of MAD2L2 mRNA in the salivary gland, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination. Fig.16C: Control (sense, Seq ID: 6718) hybridization of an adjacent section comparable to Fig.16B. Abbreviations: Br - brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; Lu - lung; SG - salivary gland; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense. Magnification x 2.7.
[00061] Figure 17. MAD2L2 expression in the adult mouse tissue arrays. Fig. 17A) X-ray film autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. High expression sites are evident in the ovary and testis and in the uterine tissue at gestation stages day 5.5 and 7.5. Fig. 17B) MAD2L2 mRNA shown in the general tissue array (TA). Low to medium level expression levels are evident in most tissue including brain, adrenal gland, spleen, thymus and liver. High MAD2L2 expression levels are confirmed in the testis and ovary. Fig. 17C) MAD2L2 mRNA in the brain tissue arrays. Expression is evident in the choroids plexus within lllrd ventricle. Fig. 17D) Control (sense, Seq ID: 6718) hybridization of the section comparable to B. Abbreviations: Adr - adrenal gland; Br - brain; ChPI - choroids plexus; K - kidney; Li - liver; OL - olfactory lobe; Ov - ovary; Te - testis; Th - thymus; UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense. Magnification x 1.6. [00062] Figure 18. MAD2L2 expression in the adult mouse testis. Fig. 18A) Emulsion autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA labeling in the wall of the seminiferous tubules seen as dark silver grains under lightfield illumination; cresyl violet staining of cell nuclei. By topography, the labeled cells may be identified a spermatocytes. Spermatogonia and spermatozoa appears as unlabeled. There is no labeling in the interstitial space Leydig cells. Fig. 18B) Control (sense, Seq ID: 6718) hybridization of an adjacent section comparable to A. Abbreviations: IS - interstitial space; Sc -spermatocyte; SfT - seminiferous tubule; Sg - spermatogonia; Sz - spermatozoa; (s) - sense. Magnification x 425.
[00063] Figure 19. MAD2L2 expression in the pregnant female uterus. Fig. 19A) Emulsion autoradiography, following hybridization with antisense (Seq ID: 6719) riboprobe, showing MAD2L2 mRNA labeling in the uterus on day 7.5 post coitum. Silver labeling is seen as dark under lightfield illumination; cresyl violet staining of cell nuclei. By topography, the labeled cells may be identified as endometrial cells. Peripherally located myometrium seems to be free of labeling. Fig. 19B) MAD2L2 labeling in the endometrium region with high PCNA activity (not shown). Fig. 19C) Fragment of the deciduas with unlabeled giant cells. Fig. 19D) Control (sense, Seq ID: 6718) hybridization of an adjacent section comparable to A. Abbreviations: G - giant cells; E - endometrium stroma cells; M - myometrium muscle cells layer; (s) - sense. Magnification x 304.
[00064] Figure 20. MCM3AP expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice. Figs. 20A-D) X-ray film autoradiography following hybridization with antisense riboprobe (Seq ID: 6721) after 5-day exposure, showing a pattern of MCM3AP mRNA distribution seen as bright labeling on darkfield. Fig. 20E) Control (sense, Seq ID: 6720) hybridization of the section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; K - kidney; Re - retina; Sp - spleen; Th - thymus; (s) - sense. Magnification x 1.6.
[00065] Figure 21. MCM3AP expression in the adult mouse. Fig. 21A) Anatomical view of the adult mouse after staining with cresyl violet. Fig. 21 B) X- ray film autoradiography after hybridization with antisense riboprobe (Seq ID: 6721) showing the presence of MCM3AP mRNA in the brain, skin, lymph node, thymus, spleen, liver, stomach, kidney and large intestine, seen as bright labeling under darkfield illumination. Fig. 21C) Control (sense, Seq ID: 6720) hybridization of an adjacent section comparable to B. Abbreviations: BM - bone marrow; Br - brain; Cb - cerebellum; H - heart; K - kidney; Li - liver; LI - large intestine; LN - lymph node; Lu - lung; SG - salivary gland; SI - small intestine; Sk - skin; Sp - spleen; St - stomach; Th - thymus; (as) - antisense; (s) - sense. Magnification x 2.7.
[00066] Figure 22. MCM3AP expression in the adult mouse tissue arrays. Fig. 22A) X-ray film autoradiography, after hybridization with antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Overall low mRNA concentration is evident. Fig. 22B) MCM3AP mRNA shown in the general tissue array (TA). MCM3AP expression levels are at the limit of the detection by ISH in most tissues including the brain, trigeminal ganglion, adrenal gland and spleen. Slightly elevated mRNA concentrations occur in the thymus. Fig. 22C) MCM3AP mRNA in the brain tissue arrays; low-level mRNA concentrations are evident in the olfactory lobe, hippocampus, hypothalamus and cerebellum. Fig. 22D) Control (sense, Seq ID: 6720) hybridization of the section comparable to B. Abbreviations: Adr - adrenal gland; Br - brain; Hip - hippocampus; Cb - cerebellum; OL - olfactory lobe; Ov - ovary; SG - salivary gland; Sp - spleen; Te - testis; Th - thymus; Ut - uterus; UtO - uterus at day 0; Ut5.5 (Ut7.5) - uterus at gestation day 5.5 (and 7.5); (s) - sense. Magnification x 1.6.
[00067] Figure 23. MCM3AP expression in the adult mouse brain hippocampus and cerebellum. Fig. 23A) Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6721), showing MCM3AP mRNA labeling (arrow) in the hippocampus area CA1 seen as bright labeling under darkfield illumination. Fig. 23B) The same fragment of the hippocampus seen under brightfield illumination. Staining tissue with cresyl violet reveals a high density of labeled cell layer. Fig. 23C) Control (sense, Seq ID: 6720) hybridization of an adjacent section comparable to A under darkfield. Fig. 23D) The same section under brightfield illumination. Abbreviations: CA1 - cornu Ammonis area 1 ; (as) - antisense; (s) - sense. Magnification: x 192.
[00068] Figure 24. NRXN1 expression in the embryonic (e10.5, e12.5 and e15.5) and postnatal (p1 and p10) mice. Figs. 24A-D) X-ray film autoradiography following hybridization with antisense Seq ID: 6723 riboprobe after 2-day exposure, showing a pattern of NRXN1 mRNA distribution seen as bright labeling on dark field. Fig. 24E) Control (sense, Seq ID: 6722) hybridization of the section comparable to D. Abbreviations: Br - brain; Cb - cerebellum; DRG - dorsal root ganglia; SC - spinal cord; (s) - sense. Magnification x 1.6.
[00069] Figure 25. NRXN 1 expression in the adult mouse. Fig. 25A) Anatomical view of the adult mouse after staining with cresyl violet. Fig. 25B) X-ray film autoradiography after hybridization with antisense riboprobe (Seq ID: 6723) showing the presence of NRXN1 mRNA in the brain, spinal cord, dorsal root ganglia and trigeminal ganglion. Fig. 25C) Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to B. Abbreviations: Br - brain; Cb - cerebellum; Cx - cortex; DRG - dorsal root ganglia; H - heart; SC - spinal cord; Sk - skin; St - stomach; TG - trigeminal ganglion; Th - thymus; (as) - antisense; (s) - sense. Magnification x 2.7.
[00070] Figure 26. NRXN1 expression in the adult mouse tissue arrays. Fig. 26A) Two-day X-ray film autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA distribution in the reproductive organs (RO) seen as bright labeling on dark field. Overall low mRNA concentration is evident. Fig. 26B) NRXN 1 mRNA shown in the general tissue array (TA). NRXN 1 expression is detectable in the CNS (brain), PNS (trigeminal ganglion) and endocrine glands (pituitary and adrenals). Fig. 26C) NRXN 1 mRNA in the brain tissue arrays. Medium to high level mRNA concentration with exception of the striatum. Fig. 26D) Control (sense, Seq ID: 6722) hybridization of the section comparable to B. Abbreviations: Adr - adrenal gland; Br - brain; Cb - cerebellum; CxVIb - cortex, deep layer VIb; Hip - hippocampus; Hy - hypothalamus; Str - striatum; Th - thalamus; (s) - sense. Magnification x 1.6.
[00071] Figure 27. NRXN1 expression in the adult mouse CNS hippocampus, cortex and PNS trigeminal ganglion. Fig. 27A) Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the cortex and hippocampus area CA1 seen as bright on darkfield illumination. Note strongly labeled deep cortical sub layer VIb. Fig. 27B) Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination. Fig. 27C) Fragment of the trigeminal ganglion seen under brightfield illumination. Fig. 27D) Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to C. Fig. 27E) Cerebral cortex at higher magnification. Large arrow indicates a labeled neuron. Thin arrow points an unlabeled presumptive glial cell. Fig. 27F) Trigeminal ganglion at higher magnification. Large arrows indicate the sensory neurons, labeled. Thin arrows point the unlabeled satellite cells. Schwann cell seen in the nerve tissue appear unlabeled. Magnifications: (A to D) x 60; (E and F) x 250.
[00072] Figure 28. NRXN1 expression in the newborn (p1) mouse PNS sensory dorsal root ganglion and ortosympathetic paravertebral ganglion. Fig. 28A) Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the dorsal root ganglion and paravertebral ganglion on darkfield illumination. Fig. 28B) The same section seen under brightfield illumination. Fig. 28C) Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination. D) The same section seen under brightfield illumination. Abbreviations: DRG - dorsal root ganglion; PVG - paravertebral ganglion; Ve - vertebrae; (as) - antisense; (s) - sense. Magnification x 60.
[00073] Figure 29. NRXN 1 expression in the postnatal and adult mouse PNS visceral Auerbach plexus. Fig. 29A) Emulsion autoradiography, after hybridization with antisense riboprobe (Seq ID: 6723), showing NRXN1 mRNA labeling in the intestine of p10 mouse. Arrows indicate group of neurons present in the smooth muscle cell layer. Fig. 29B) The same section seen under brightfield illumination. Fig. 29C) Control (sense, Seq ID: 6722) hybridization of an adjacent section comparable to A under darkfield illumination. Fig. 29D) The same section seen under brightfield illumination. Fig. 29E) NRXN1 mRNA-labeled neuron in the Auerbach plexus (arrows) in the postnatal mouse intestine found in the space between circular and longitudinal smooth muscles layer showing . Fig. 29F) NRXN1 mRNA-labeled neuron in Auerbach plexus in the adult mouse intestine showing an inferior labeling intensity when compared to that of p10 mouse plexus. Abbreviations: M - smooth muscle fibers; MC - circular musclular layer; ML - longitudinal muscular layer; V - intestinal villi; (as) - antisense; (s) - sense. Magnifications: (A to D) x 60; (E and F) x 250.
[00074] TABLE DESCRIPTION
[00075] Table 1. List of Endometriosis disease candidate regions identified from the Genome Wide Scan association analyses. The first column denotes the region identifier. The second and third columns correspond to the chromosome and cytogenetic band, respectively. The fourth and fifth columns correspond to the chromosomal start and end coordinates of the NCBI genome assembly derived from build 36.
[00076] Table 2. List of candidate genes from the regions identified from the genome wide association analysis. The first column corresponds to the region identifier provided in Table 1. The second and third columns correspond to the chromosome and cytogenetic band, respectively. The fourth and fifth columns corresponds to the chromosomal start coordinates of the NCBI genome assembly derived from build 36 (B36) and the end coordinates (the start and end position relate to the + orientation of the NCBI assembly and don't necessarily correspond to the orientation of the gene). The sixth and seventh columns correspond to the official gene symbol and gene name, respectively, and were obtained from the NCBI Entrez Gene database. The eighth column corresponds to the NCBI Entrez Gene Identifier (GenelD). The ninth and tenth columns correspond to the Sequence IDs from nucleotide (cDNA) and protein entries in the Sequence Listing.
[00077] Table 3. List of candidate genes based on EST clustering from the regions identified from the various genome wide analyses. The first column corresponds to the region identifier provided in Table 1. The second column corresponds to the chromosome number. The third and fourth columns correspond to the chromosomal start and end coordinates of the NCBI genome assemblies derived from build 36 (B36). The fifth column corresponds to the ECGene Identifier, corresponding to the ECGene track of UCSC. These ECGene entries were determined by their overlap with the regions from Table 1 , based on the start and end coordinates of both Region and ECGene identifiers. The sixth and seventh columns correspond to the Sequence IDs from nucleotide and protein entries in the Sequence Listing.
[00078] Table 4. List of micro RNA (miRNA) from the regions identified from the genome wide association analyses derived from build 36 (B36). To identify the miRNA from B36, these miRNA entries were determined by their overlap with the regions from Table 1 , based on the start and end coordinates of both Region and miRNA identifiers. The first column corresponds to the region identifier provided in Table 1. The second column corresponds to the chromosome number. The third and fourth columns correspond to the chromosomal start and end coordinates of the NCBI genome assembly derived from build 36 (the start and end position relate to the + orientation of the NCBI assembly and do not necessarily correspond to the orientation of the miRNA). The fifth and sixth columns correspond to the miRNA accession and miRNA id, respectively, and were obtained from the miRBase database. The seventh column corresponds to the NCBI Entrez Gene Identifier (GenelD). The eighth column corresponds to the Sequence ID from nucleotide (RNA) in the Sequence Listing.
[00079] Table 5.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: GWS win1. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00080] Table 5. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 5.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00081] Table 6.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: HASJNFERTILE. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, -Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00082] Table 6.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 6.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00083] Table 7.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: NOT INFERTILE. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - log 10 P values for GWS, - log 10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00084] Table 7.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 7.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00085] Table 8.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: NOT PELVIC PAIN. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00086] Table 8.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 8.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00087] Table 9.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: HAS OVARIAN CYST. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00088] Table 9.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 9.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00089] Table 10.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: NOT OVARYAN CYSTS. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes. [00090] Table 10.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 10.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00091] Table 11.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: Stage 3 & 4. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - log 10 P values for GWS, - log 10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00092] Table 11.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 11.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00093] Table 12.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: has PRKCE- 1-1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00094] Table 12.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 12.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00095] Table 13.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: not_RAF- 1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00096] Table 13.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 13.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00097] Table 14.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: has_DNAH5- 1_cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[00098] Table 14.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 14.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[00099] Table 15.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: has SYNE1-1 cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[000100] Table 15.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 15.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[000101] Table 16.1. Genome wide association study results in the Quebec Founder Population (QFP). SNP markers found to be associated with Endometriosis from the analysis of genome wide scan (GWS) data: not SYNE1-1 cr. Columns include: Region ID; Chromosome; Build 36 location in base pairs (bp); rs#, dbSNP data base (NCBI) reference number; Sequence ID, unique numerical identifier for this patent application; Sequence, 21 bp of sequence covering 10 base pair of unique sequence flanking either side of central polymorphic SNP; - Iog10 P values for GWS, - Iog10 of the P value for statistical significance from the GWS for single SNP markers (both T test and Permutation test p-values are displayed; see Example section) and for the most highly associated multi-marker haplotypes centered at the reference marker and defined by the sliding windows of specified sizes.
[000102] Table 16.2. List of significantly associated haplotypes based on the Endometriosis Disease results using the Quebec Founder Population (QFP). Individual haplotypes with associated relative risks are presented in each row of the table; these values were extracted from the associated marker haplotype window with the most significant p value for each SNP in Table 16.1. The first column lists the region ID as presented in Table 1. The Haplotype column lists the specific nucleotides for the individual SNP alleles contributing to the haplotype reported. The Case and Control columns correspond to the numbers of cases and controls, respectively, containing the haplotype variant noted in the Haplotype column. The Total Case and Total Control columns list the total numbers of cases and controls for which genotype data was available for the haplotype in question. The RR column gives to the relative risk for each particular haplotype. The remainder of the columns lists the SeqIDs for the SNPs contributing to the haplotype and their relative location with respect to the central marker. The Central marker (0) column lists the SeqlD for the central marker on which the haplotype is based. Flanking markers are identified by minus (-) or plus (+) signs to indicate the relative location of flanking SNPs.
[000103] Table 17. Description of primer sequences used for the semiquantitative gene expression profiling by RT-PCR (see Example section for details).
[000104] Table 18. Probes used for the in situ hybridization (ISH) study (see Example section for details).
DEFINITIONS
[000105] Throughout the description of the present invention, several terms are used that are specific to the science of this field. For the sake of clarity and to avoid any misunderstanding, these definitions are provided to aid in the understanding of the specification and claims.
[000106] Allele: One of a pair, or series, of forms of a gene or non-genic region that occur at a given locus in a chromosome. Alleles are symbolized with the same basic symbol (e.g., B for dominant and b for recessive; B1 , B2, Bn for n additive alleles at a locus). In a normal diploid cell there are two alleles of any one gene (one from each parent), which occupy the same relative position (locus) on homologous chromosomes. Within a population there may be more than two alleles of a gene. See multiple alleles. SNPs also have alleles, i.e., the two (or more) nucleotides that characterize the SNP.
[000107] Amplification of nucleic acids: refers to methods such as polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known in the art and are described, for example, in U.S. Patent Nos. 4,683,195 and 4,683,202. Reagents and hardware for conducting PCR are commercially available. Primers useful for amplifying sequences from the disorder region are preferably complementary to, and preferably hybridize specifically to, sequences in the disorder region or in regions that flank a target region therein. Genes from Tables 2-4 generated by amplification may be sequenced directly. Alternatively, the amplified sequence(s) may be cloned prior to sequence analysis.
[000108] Antigenic component: is a moiety that binds to its specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
[000109] Antibodies: refer to polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, that can bind to proteins and fragments thereof or to nucleic acid sequences from the disorder region, particularly from the disorder gene products or a portion thereof. The term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Proteins may be prepared synthetically in a protein synthesizer and coupled to a carrier molecule and injected over several months into rabbits. Rabbit sera are tested for immunoreactivity to the protein or fragment. Monoclonal antibodies may be made by injecting mice with the proteins, or fragments thereof. Monoclonal antibodies can be screened by ELISA and tested for specific immunoreactivity with protein or fragments thereof (Harlow et al. 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). These antibodies will be useful in developing assays as well as therapeutics.
[000110] Associated allele: refers to an allele at a polymorphic locus that is associated with a particular phenotype of interest, e.g., a predisposition to a disorder or a particular drug response.
[000111] cDNA: refers to complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus, a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, included in a cloning vector or PCR amplified. This term includes genes from which the intervening sequences have been removed.
[000112] cDNA library: refers to a collection of recombinant DNA molecules containing cDNA inserts that together comprise essentially all of the expressed genes of an organism or tissue. A cDNA library can be prepared by methods known to one skilled in the art (see, e.g., Cowell and Austin, 1997, "DNA Library Protocols," Methods in Molecular Biology). Generally, RNA is first isolated from the cells of the desired organism, and the RNA is used to prepare cDNA molecules.
[000113] Cloning: refers to the use of recombinant DNA techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, it is necessary to use methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
[000114] Cloning vector: refers to a plasmid or phage DNA or other DNA molecule that is able to replicate in a host cell. The cloning vector is typically characterized by one or more endonuclease recognition sites at which such DNA sequences may be cleaved in a determinable fashion without loss of an essential biological function of the DNA, and which may contain a selectable marker suitable for use in the identification of cells containing the vector.
[000115] Coding sequence or a protein-coding sequence: is a polynucleotide sequence capable of being transcribed into mRNA and/or capable of being translated into a polypeptide or peptide. The boundaries of the coding sequence are typically determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. [000116] Complement of a nucleic acid sequence: refers to the antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
[000117] Disorder region: refers to the portions of the human chromosomes displayed in Table 1 bounded by the markers from Tables 2-16.
[000118] Disorder-associated nucleic acid or polypeptide sequence: refers to a nucleic acid sequence that maps to region of Table 1 or the polypeptides encoded therein (Tables 2-4, nucleic acids, and polypeptides). For nucleic acids, this encompasses sequences that are identical or complementary to the gene sequences from Tables 2-4, as well as sequence-conservative, function- conservative, and non-conservative variants thereof. For polypeptides, this encompasses sequences that are identical to the polypeptide, as well as function-conservative and non-conservative variants thereof. Included are the alleles of naturally-occurring polymorphisms causative of ENDOMETRIOSIS disease such as, but not limited to, alleles that cause altered expression of genes of Tables 2-4 and alleles that cause altered protein levels or stability (e.g., decreased levels, increased levels, expression in an inappropriate tissue type, increased stability, and decreased stability).
[000119] Expression vector: refers to a vehicle or plasmid that is capable of expressing a gene that has been cloned into it, after transformation or integration in a host cell. The cloned gene is usually placed under the control of (i.e., operably linked to) a regulatory sequence.
[000120] Function-conservative variants: are those in which a change in one or more nucleotides in a given codon position results in a polypeptide sequence in which a given amino acid residue in the polypeptide has been replaced by a conservative amino acid substitution. Function-conservative variants also include analogs of a given polypeptide and any polypeptides that have the ability to elicit antibodies specific to a designated polypeptide. [000121] Founder population: Also a population isolate, this is a large number of people who have mostly descended, in genetic isolation from other populations, from a much smaller number of people who lived many generations ago.
[000122] Gene: Refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term "gene" also refers to a DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions, as well as regulatory regions, and can include 5' and 3' ends. A gene sequence is wild-type if such sequence is usually found in individuals unaffected by the disorder or condition of interest. However, environmental factors and other genes can also play an important role in the ultimate determination of the disorder. In the context of complex disorders involving multiple genes (oligogenic disorder), the wild type, or normal sequence can also be associated with a measurable risk or susceptibility, receiving its reference status based on its frequency in the general population.
[000123] GeneMaps: are defined as groups of gene(s) that are directly or indirectly involved in at least one phenotype of a disorder (some non-limiting example of GeneMaps comprises varius combinations of genes from Tables 2-4). As such, GeneMaps enable the development of synergistic diagnostic products, creating "theranostics".
[000124] Genotype: Set of alleles at a specified locus or loci.
[000125] Haplotype: The allelic pattern of a group of (usually contiguous) DNA markers or other polymorphic loci along an individual chromosome or double helical DNA segment. Haplotypes identify individual chromosomes or chromosome segments. The presence of shared haplotype patterns among a group of individuals implies that the locus defined by the haplotype has been inherited, identical by descent (IBD), from a common ancestor. Detection of identical by descent haplotypes is the basis of linkage disequilibrium (LD) mapping. Haplotypes are broken down through the generations by recombination and mutation. In some instances, a specific allele or haplotype may be associated with susceptibility to a disorder or condition of interest, e.g., ENDOMETRIOSIS disease. In other instances, an allele or haplotype may be associated with a decrease in susceptibility to a disorder or condition of interest, i.e., a protective sequence.
[000126] Host: includes prokaryotes and eukaryotes. The term includes an organism or cell that is the recipient of an expression vector (e.g., autonomously replicating or integrating vector).
[000127] Hybridizable: nucleic acids are hybridizable to each other when at least one strand of the nucleic acid can anneal to another nucleic acid strand under defined stringency conditions. In some embodiments, hybridization requires that the two nucleic acids contain at least 10 substantially complementary nucleotides; depending on the stringency of hybridization, however, mismatches may be tolerated. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, and can be determined in accordance with the methods described herein.
[000128] Identity by descent (IBD): Identity among DNA sequences for different individuals that is due to the fact that they have all been inherited from a common ancestor. LD mapping identifies IBD haplotypes as the likely location of disorder genes shared by a group of patients.
[000129] Identity: as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity can be readily calculated by known methods, including but not limited to those described in A.M. Lesk (ed), 1988, Computational Molecular Biology, Oxford University Press, NY; D.W. Smith (ed), 1993, Biocomputing. Informatics and Genome Projects, Academic Press, NY; A.M. Griffin and H. G. Griffin, H. G (eds), 1994, ComputerAnalysis of Sequence Data, Part 1 , Humana Press, NJ; G. von Heinje, 1987, Sequence Analysis in Molecular Biology, Academic Press; and M. Gribskov and J. Devereux (eds), 1991 , Sequence Analysis Primer, M Stockton Press, NY; H. Carillo and D. Lipman, 1988, SIAM J. Applied Math., 48:1073.
[000130] Immunogenic component: is a moiety that is capable of eliciting a humoral and/or cellular immune response in a host animal.
[000131] Isolated nucleic acids: are nucleic acids separated away from other components (e.g., DNA, RNA, and protein) with which they are associated (e.g., as obtained from cells, chemical synthesis systems, or phage or nucleic acid libraries). Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components. In accordance with the present invention, isolated nucleic acids can be obtained by methods described herein, or other established methods, including isolation from natural sources (e.g., cells, tissues, or organs), chemical synthesis, recombinant methods, combinations of recombinant and chemical methods, and library screening methods.
[000132] Isolated polypeptides or peptides: are those that are separated from other components (e.g., DNA, RNA, and other polypeptides or peptides) with which they are associated (e.g., as obtained from cells, translation systems, or chemical synthesis systems). In a preferred embodiment, isolated polypeptides or peptides are at least 10% pure; more preferably, 80% or 90% pure. Isolated polypeptides and peptides include those obtained by methods described herein, or other established methods, including isolation from natural sources (e.g., cells, tissues, or organs), chemical synthesis, recombinant methods, or combinations of recombinant and chemical methods. Proteins or polypeptides referred to herein as recombinant are proteins or polypeptides produced by the expression of recombinant nucleic acids. A portion as used herein with regard to a protein or polypeptide, refers to fragments of that protein or polypeptide. The fragments can range in size from 5 amino acid residues to all but one residue of the entire protein sequence. Thus, a portion or fragment can be at least 5, 5-50, 50-100, I00-200, 200-400, 400-800, or more consecutive amino acid residues of a protein or polypeptide.
[000133] Linkage disequilibrium (LD): the situation in which the alleles for two or more loci do not occur together in individuals sampled from a population at frequencies predicted by the product of their individual allele frequencies. In other words, markers that are in LD do not follow Mendel's second law of independent random segregation. LD can be caused by any of several demographic or population artifacts as well as by the presence of genetic linkage between markers. However, when these artifacts are controlled and eliminated as sources of LD, then LD results directly from the fact that the loci involved are located close to each other on the same chromosome so that specific combinations of alleles for different markers (haplotypes) are inherited together. Markers that are in high LD can be assumed to be located near each other and a marker or haplotype that is in high LD with a genetic trait can be assumed to be located near the gene that affects that trait. The physical proximity of markers can be measured in family studies where it is called linkage or in population studies where it is called linkage disequilibrium.
[000134] LD mapping: population based gene mapping, which locates disorder genes by identifying regions of the genome where haplotypes or marker variation patterns are shared statistically more frequently among disorder patients compared to healthy controls. This method is based upon the assumption that many of the patients will have inherited an allele associated with the disorder from a common ancestor (IBD), and that this allele will be in LD with the disorder gene.
[000135] Locus: a specific position along a chromosome or DNA sequence. Depending upon context, a locus could be a gene, a marker, a chromosomal band or a specific sequence of one or more nucleotides. [000136] Minor allele frequency (MAF): the population frequency of one of the alleles for a given polymorphism, which is equal or less than 50%. The sum of the MAF and the Major allele frequency equals one.
[000137] Markers: an identifiable DNA sequence that is variable (polymorphic) for different individuals within a population. These sequences facilitate the study of inheritance of a trait or a gene. Such markers are used in mapping the order of genes along chromosomes and in following the inheritance of particular genes; genes closely linked to the marker or in LD with the marker will generally be inherited with it. Two types of markers are commonly used in genetic analysis, microsatellites and SNPs.
[000138] Microsatellite: DNA of eukaryotic cells comprising a repetitive, short sequence of DNA that is present as tandem repeats and in highly variable copy number, flanked by sequences unique to that locus.
[000139] Mutant sequence: if it differs from one or more wild-type sequences. For example, a nucleic acid from a gene listed in Tables 2-4 containing a particular allele of a single nucleotide polymorphism may be a mutant sequence. In some cases, the individual carrying this allele has increased susceptibility toward the disorder or condition of interest. In other cases, the mutant sequence might also refer to an allele that decreases the susceptibility toward a disorder or condition of interest and thus acts in a protective manner. The term mutation may also be used to describe a specific allele of a polymorphic locus.
[000140] Non-conservative variants: are those in which a change in one or more nucleotides in a given codon position results in a polypeptide sequence in which a given amino acid residue in a polypeptide has been replaced by a non- conservative amino acid substitution. Non-conservative variants also include polypeptides comprising non-conservative amino acid substitutions.
[000141] Nucleic acid or polynucleotide: purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo polydeoxyribonucleotides. This includes single-and double- stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as protein nucleic acids (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
[000142] Nucleotide: a nucleotide, the unit of a DNA molecule, is composed of a base, a 2'-deoxyribose and phosphate ester(s) attached at the 5' carbon of the deoxyribose. For its incorporation in DNA, the nucleotide needs to possess three phosphate esters but it is converted into a monoester in the process.
[000143] Operably linked: means that the promoter controls the initiation of expression of the gene. A promoter is operabiy linked to a sequence of proximal DNA if upon introduction into a host cell the promoter determines the transcription of the proximal DNA sequence(s) into one or more species of RNA. A promoter is operably linked to a DNA sequence if the promoter is capable of initiating transcription of that DNA sequence.
[000144] Ortholog: denotes a gene or polypeptide obtained from one species that has homology to an analogous gene or polypeptide from a different species.
[000145] Paralog: denotes a gene or polypeptide obtained from a given species that has homology to a distinct gene or polypeptide from that same species.
[000146] Phenotype: any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to, a disorder.
[000147] Polymorphism: occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals at a single locus. A polymorphic site thus refers specifically to the locus at which the variation occurs. In some cases, an individual carrying a particular allele of a polymorphism has an increased or decreased susceptibility toward a disorder or condition of interest.
[000148] Portion and fragment: are synonymous. A portion as used with regard to a nucleic acid or polynucleotide refers to fragments of that nucleic acid or polynucleotide. The fragments can range in size from 8 nucleotides to all but one nucleotide of the entire gene sequence. Preferably, the fragments are at least about 8 to about 10 nucleotides in length; at least about 12 nucleotides in length; at least about 15 to about 20 nucleotides in length; at least about 25 nucleotides in length; or at least about 35 to about 55 nucleotides in length.
[000149] Probe or primer: refers to a nucleic acid or oligonucleotide that forms a hybrid structure with a sequence in a target region of a nucleic acid due to complementarity of the probe or primer sequence to at least one portion of the target region sequence.
[000150] Protein and polypeptide: are synonymous. Peptides are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity (e.g., proteolysis, adhesion, fusion, antigenic, or intracellular activity) as the complete polypeptide sequence.
[000151] Recombinant nucleic acids: nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. Portions of recombinant nucleic acids which code for polypeptides can be identified and isolated by, for example, the method of M. Jasin et al., U.S. Patent No. 4,952,501.
[000152] Regulatory sequence: refers to a nucleic acid sequence that controls or regulates expression of structural genes when operably linked to those genes. These include, for example, the lac systems, the trp system, major operator and promoter regions of the phage lambda, the control region of fd coat protein and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells. Regulatory sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host, and may contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements and/or translational initiation and termination sites. [000153] Sample: as used herein refers to a biological sample, such as, for example, tissue or fluid isolated from an individual or animal (including, without limitation, plasma, serum, cerebrospinal fluid, lymph, tears, nails, hair, saliva, milk, pus, and tissue exudates and secretions) or from in vitro cell culture- constituents, as well as samples obtained from, for example, a laboratory procedure.
[000154] Single nucleotide polymorphism (SNP): variation of a single nucleotide. This includes the replacement of one nucleotide by another and deletion or insertion of a single nucleotide. Typically, SNPs are biallelic markers although tri- and tetra-allelic markers also exist. For example, SNP A\C may comprise allele C or allele A (Tables 5-16). Thus, a nucleic acid molecule comprising SNP A\C may include a C or A at the polymorphic position. For clarity purposes, an ambiguity code is used in Tables 5-16 and the sequence listing, to represent the variations. For a combination of SNPs, the term "haplotype" is used, e.g. the genotype of the SNPs in a single DNA strand that are linked to one another. In certain embodiments, the term "haplotype" is used to describe a combination of SNP alleles, e.g., the alleles of the SNPs found together on a single DNA molecule. In specific embodiments, the SNPs in a haplotype are in linkage disequilibrium with one another.
[000155] Sequence-conservative: variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position (i.e., silent mutation).
[000156] Substantially homologous: a nucleic acid or fragment thereof is substantially homologous to another if, when optimally aligned (with appropriate nucleotide insertions and/or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least 60% of the nucleotide bases, usually at least 70%, more usually at least 80%, preferably at least 90%, and more preferably at least 95-98% of the nucleotide bases. Alternatively, substantial homology exists when a nucleic acid or fragment thereof will hybridize, under selective hybridization conditions, to another nucleic acid (or a complementary strand thereof). Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Typically, selective hybridization will occur when there is at least about 55% sequence identity over a stretch of at least about nine or more nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% (M. Kanehisa, 1984, NucL Acids Res. 11 :203-213). The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least 14 nucleotides, usually at least 20 nucleotides, more usually at least 24 nucleotides, typically at least 28 nucleotides, more typically at least 32 nucleotides, and preferably at least 36 or more nucleotides.
[000157] Wild-type gene from Tables 2-4: refers to the reference sequence. The wild-type gene sequences from Tables 2-4 used to identify the variants (polymorphisms, alleles, and haplotypes) described in detail herein.
[000158] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies known to those of skill in the art. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of recombinant DNA technology include J. Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; P. B. Kaufman et al., (eds), 1995, Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton; M.J. McPherson (ed), 1991 , Directed Mutagenesis: A Practical Approach, IRL Press, Oxford; J. Jones, 1992, Amino Acid and Peptide Synthesis, Oxford Science Publications, Oxford; B. M. Austen and O. M. R. Westwood, 1991 , Protein Targeting and Secretion, IRL Press, Oxford; D.N Glover (ed), 1985, DNA Cloning, Volumes I and 11 ; MJ. Gait (ed), 1984, Oligonucleotide Synthesis; B. D. Hames and S.J. Higgins (eds), 1984, Nucleic Acid Hybridization; Quirke and Taylor (eds), 1991 , PCR-A Practical Approach; Harries and Higgins (eds), 1984, Transcription and Translation; R.I. Freshney (ed), 1986, Animal Cell Culture; Immobilized Cells and Enzymes, 1986, IRL Press; Perbal, 1984, A Practical Guide to Molecular Cloning, J. H. Miller and M. P. Calos (eds), 1987, Gene Transfer Vectors for Mammalian Cells, Cold Spring Harbor Laboratory Press; MJ. Bishop (ed), 1998, Guide to Human Genome Computing, 2d Ed., Academic Press, San Diego, CA; L.F. Peruski and A.H. Peruski, 1997, The Internet and the New Biology. Tools for Genomic and Molecular Research, American Society for Microbiology, Washington, D. C. Standard reference works setting forth the general principles of immunology include S. Sell, 1996, Immunology, immunopathology & Immunity, 5th Ed., Appleton & Lange, Publ., Stamford, CT; D. Male et a/., 1996, Advanced Immunology, 3d Ed., Times Mirror Int'l Publishers Ltd., Publ., London; D. P. Stites and A.L Terr, 1991 , Basic and Clinical Immunology, 7th Ed., Appleton & Lange, Publ., Norwalk, CT; and A.K. Abbas et al., 1991 , Cellular and Molecular Immunology, W. B. Saunders Co., Publ., Philadelphia, PA. Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention; however, preferred materials and/or methods are described. Materials, reagents, and the like to which reference is made in the following description and examples are generally obtainable from commercial sources, and specific vendors are cited herein.
DETAILED DESCRIPTION OF THE INVENTION
Genome wide association study to construct a GeneMap for ENDOMETRIOSIS
[000159] The present invention is based on the discovery of genes associated with ENDOMETRIOSIS disease. In the preferred embodiment, disease- associated loci (candidate regions; Table 1) are identified by the statistically significant differences in allele or haplotype frequencies between the cases and the controls. For the purpose of the present invention candidate regions are identified in Table 1. [000160] The invention provides a method for the discovery of genes associated with ENDOMETRIOSIS disease and the construction of a GeneMap for ENDOMETRIOSIS disease in a human population, comprising the following steps (see also Example section herein). The GeneMaps of the invention, presented in the Example section, is provided for clarity purposes and other GeneMaps with various other combinations of genes from Tables 2-4 and/or other genes involved in the related networks or pathways are obtained by the methods of the invention:
[000161] Step 1 : Recruit patients (cases) and controls
[000162] In the preferred embodiment, 500 patients diagnosed for ENDOMETRIOSIS disease along with 500 independent controls samples are recruited from the Quebec Founder Population (QFP).
[000163] In another embodiment, more or less than 500 patients and controls are recruited.
[000164] In another embodiment, 500 patients diagnosed for ENDOMETRIOSIS disease along with two family members are recruited from the Quebec Founder Population (QFP). The preferred trios recruited are parent- parent-child (PPC) trios. Trios can also be recruited as parent-child-child (PCC) trios. In another preferred embodiment, more or less than 500 trios are recruited
[000165] In yet another embodiment, the present invention is performed as a whole or partially with DNA samples from individuals of another founder population than the Quebec population or from the general population.
[000166] Step 2: DNA extraction and quantitation
[000167] Any sample comprising cells or nucleic acids from patients or controls may be used. Preferred samples are those easily obtained from the patient or control. Such samples include, but are not limited to blood, peripheral lymphocytes, buccal swabs, epithelial cell swabs, nails, hair, bronchoalveolar lavage fluid, sputum, or other body fluid or tissue obtained from an individual. [000168] In one embodiment, DNA is extracted from such samples in the quantity and quality necessary to perform the invention using conventional DNA extraction and quantitation techniques. The present invention is not linked to any DNA extraction or quantitation platform in particular.
[000169] Step 3: Genotype the recruited individuals
[000170] In one embodiment, assay-specific and/or locus-specific and/or allele- specific oligonucleotides for every SNP marker of the present invention (Tables 5-16) are organized onto one or more arrays. The genotype at each SNP locus is revealed by hybridizing short PCR fragments comprising each SNP locus onto these arrays. The arrays permit a high-throughput genome wide association study using DNA samples from individuals of the Quebec founder population. Such assay-specific and/or locus-specific and/or allele-specific oligonucleotides necessary for scoring each SNP of the present invention are preferably organized onto a solid support. Such supports can be arrayed on wafers, glass slides, beads or any other type of solid support.
[000171] In another embodiment, the assay-specific and/or locus-specific and/or allele-specific oligonucleotides are not organized onto a solid support but are still used as a whole, in panels or one by one. The present invention is therefore not linked to any genotyping platform in particular.
[000172] In another embodiment, one or more portions of the SNP maps (publicly available maps and our own proprietary QLDM map) are used to screen the whole genome, a subset of chromosomes, a chromosome, a subset of genomic regions or a single genomic region.
[000173] In the preferred embodiment, the individuals composing the cases and controls or the trios are preferably individually genotyped with at least 80,000 markers, generating at least a few million genotypes; more preferably, at least a hundred million. In another embodiment, individuals are pooled in cases and control pools for genotyping and genetic analysis. [000174] Step 4: Exclude the markers that did not pass the quality control of the assay.
[000175] Preferably, the quality controls comprises, but are not limited to, the following criteria: eliminate SNPs that had a high rate of Mendelian errors (cut-off at 1 % Mendelian error rate), that deviate from the Hardy-Weinberg equilibrium, that are non-polymorphic in the Quebec founder population or have too many missing data (cut-off at 1 % missing values or higher), or simply because they are non-polymorphic in the Quebec founder population (cut-off at 1 % < 10% minor allele frequency (MAF)).
[000176] Step 5: Perform the genetic analysis on the results obtained using haplotype information as well as single-marker association.
[000177] In the preferred embodiment, genetic analysis is performed on all the genotypes from Step 3.
[000178] In another embodiment, genetic analysis is performed on a subset of markers from Step 3 or from markers that passed the quality controls from Step 4.
[000179] In one embodiment, the genetic analysis consists of, but is not limited to features corresponding to Phase information and haplotype structures. Phase information and haplotype structures are preferably deduced from trio genotypes using Phasefinder. Since chromosomal assignment (phase) cannot be estimated when all trio members are heterozygous, an Expectation-Maximization (EM) algorithm may be used to resolve chromosomal assignment ambiguities after Phasefinder.
[000180] In yet another embodiment, the PL-EM algorithm (Partition-Ligation EM; Niu et a/.., Am. J. Hum. Genet. 70:157 (2002)) can be used to estimate haplotypes from the "genotype" data as a measured estimate of the reference allele frequency of a SNP in 15-marker windows that advance in increments of one marker across the data set. The results from such algorithms are converted into 15-marker haplotype files. Subsequently, the individual 15-marker block files are assembled into one continuous block of haplotypes for the entire chromosome. These extended haplotypes can then be used for further analysis. Such haplotype assembly algorithms take the consensus estimate of the allele call at each marker over all separate estimations (most markers are estimated 15 different times as the 15 marker blocks pass over their position).
[000181] In another embodiment, the haplotype frequencies among patients are compared to those among the controls using LDSTATS, a program that assesses the association of haplotypes with the disease. Such program defines haplotypes using multi-marker windows that advance across the marker map in one-marker increments. Such windows can be 1 , 3, 5, 7 or 9 markers wide, and all these window sizes are tested concurrently. Larger multi-marker haplotype windows can also be used. At each position the frequency of haplotypes in cases is compared to the frequency of haplotypes in controls. Such allele frequency differences for single marker windows can be tested using Pearson's Chi-square with any degree of freedom. Multi-allelic haplotype association can be tested using Smith's normalization of the square root of Pearson's Chi-square. Such significance of association can be reported in two ways:
[000182] The significance of association within any one haplotype window is plotted against the marker that is central to that window.
[000183] P-values of association for each specific marker are calculated as a pooled P-value across all haplotype windows in which they occur. The pooled P- value is calculated using an expected value and variance calculated using a permutation test that considers covariance between individual windows. Such pooled P-values can yield narrower regions of gene location than the window data (see Example 3 herein for details on various analysis methods, such as LDSTATS v2.0 and v4.0).
[000184] In another embodiment, conditional haplotype and subtype analyses can be performed on subsets of the original set of cases and controls using the program LDSTATS. For conditional analyses, the selection of a subset of cases and their matched controls can be based on the carrier status of cases at a gene or locus of interest (see conditional analysis section in Example 3 herein). Various conditional haplotypes can be derived, such as protective haplotypes and risk haplotypes.
[000185] Step 6: SNP and DNA polymorphism discovery
[000186] In the preferred embodiment, all the candidate genes and regions identified in step 5 are sequenced for polymorphism identification.
[000187] In another embodiment, the entire region, including all introns, is sequenced to identify all polymorphisms.
[000188] In yet another embodiment, the candidate genes are prioritized for sequencing, and only functional gene elements (promoters, conserved noncoding sequences, exons and splice sites) are sequenced.
[000189] In yet another embodiment, previously identified polymorphisms in the candidate regions can also be used. For example, SNPs from dbSNP, or others can also be used rather than resequencing the candidate regions to identify polymorphisms.
[000190] The discovery of SNPs and DNA polymorphisms generally comprises a step consisting of determining the major haplotypes in the region to be sequenced. The preferred samples are selected according to which haplotypes contribute to the association signal observed in the region to be sequenced. The purpose is to select a set of samples that covers all the major haplotypes in the given region. Each major haplotype is preferably analyzed in at least a few individuals.
[000191] Any analytical procedure may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an amplification reaction and optionally a signal generation system. Any means of mutation detection or discrimination may be used. For instance, DNA sequencing, scanning methods, hybridization, extension based methods, incorporation based methods, restriction enzyme- based methods and ligation-based methods may be used in the methods of the invention.
[000192] Sequencing methods include, but are not limited to, direct sequencing, and sequencing by hybridization. Scanning methods include, but are not limited to, protein truncation test (PTT), single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), cleavage, heteroduplex analysis, chemical mismatch cleavage (CMC), and enzymatic mismatch cleavage. Hybridization-based methods of detection include, but are not limited to, solid phase hybridization such as dot blots, multiple allele specific diagnostic assay (MASDA), reverse dot blots, and oligonucleotide arrays (DNA Chips). Solution phase hybridization amplification methods may also be used, such as Taqman. Extension based methods include, but are not limited to, amplification refraction mutation systems (ARMS), amplification refractory mutation systems (ALEX), and competitive oligonucleotide priming systems (COPS). Incorporation based methods include, but are not limited to, mini-sequencing and arrayed primer extension (APEX). Restriction enzyme-based detection systems include, but are not limited to, restriction site generating PCR. Lastly, ligation based detection methods include, but are not limited to, oligonucleotide ligation assays (OLA). Signal generation or detection systems that may be used in the methods of the invention include, but are not limited to, fluorescence methods such as fluorescence resonance energy transfer (FRET), fluorescence quenching, fluorescence polarization as well as other chemiluminescence, electrochemiluminescence, Raman, radioactivity, colometric methods, hybridization protection assays and mass spectrometry methods. Further amplification methods include, but are not limited to self sustained replication (SSR), nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and branched DNA (B-DNA). [000193] Sequencing can also be performed using a proprietary sequencing technology (Cantaloupe; PCT/EP2005/002870).
[000194] Step 7: Ultrafine Mapping
[000195] This step further maps the candidate regions and genes confirmed in the previous step to identify and validate the responsible polymorphisms associated with ENDOMETRIOSIS disease in the human population.
[000196] In a preferred embodiment, the discovered SNPs and polymorphisms of step 6 are ultrafine mapped at a higher density of markers than the GWS described herein using the same technology described in step 3.
[000197] Step 8: GeneMap construction
[000198] The confirmed variations in DNA (including both genie and non-genic regions) are used to build a GeneMap for ENDOMETRIOSIS disease. The gene content of this GeneMap is described in more detail below. Such GeneMap can be used for other methods of the invention comprising the diagnostic methods described herein, the susceptibility to ENDOMETRIOSIS disease, the response to a particular drug, the efficacy of a particular drug, the screening methods described herein and the treatment methods described herein.
[000199] As is evident to one of ordinary skill in the art, all of the above steps or the steps do not need to be performed, or performed in a given order to practice or use the SNPs, genomic regions, genes, proteins, etc. in the methods of the invention.
Genes from the GeneMap
[000200] In one embodiment the GeneMap consists of genes and targets, in a variety of combinations, identified from the candidate regions listed in Table 1. In another embodiment, all genes from Tables 2-4 are present in the GeneMap. In another preferred embodiment, the GeneMap consists of a selection of genes from Tables 2-4. The genes of the invention (Tables 2-4) are arranged by candidate regions and by their chromosomal location. Such order is for the purpose of clarity and does not reflect any other criteria of selection in the association of the genes with ENDOMETRIOSIS. In yet another embodiment, the GeneMaps of the invention consists of a selection of genes from Tables 2-4 and a selection of genes that are interactors (direct or indirect) with the genes from the Tables. For clarity purposes, those interactor genes are not present in Tables 2-4, but know in the art from various public documents (scientific articles, patent literature etc.). The GeneMaps represent the knowledge that is needed for therapeutic and diagnostic intervention for a particular disease. The GeneMaps aid in the selection of the best target to intervene in a disease state. Each disease can be subdivided into various disease states and sub-phenotypes, thus various GeneMaps are needed to address various disease sub-phenopypes, and a clinical population can be stratified by sub-phenotype, which would be covered by a particular GeneMap.
[000201] In one embodiment, genes identified in the WGAS and subsequent studies are evaluated using the Ingenuity Pathway Analysis application (IPA, Ingenuity systems) in order to identify direct biological interactions between these genes, and also to identify molecular regulators acting on those genes (indirect interactions) that could be also involved in ENDOMETRIOSIS. The purpose of this effort is to decipher the molecules involved in contributing to ENDOMETRIOSIS. These gene interaction networks are very valuable tools in the sense that they facilitate extension of the map of gene products that could represent potential drug targets for ENDOMETRIOSIS.
[000202] In another embodiment, other means (such as fuctional biochemical assays and genetic asssays) are used to identify the biological interactions between genes to create a GeneMap (see Example section herein for description of the various GeneMaps).
Nucleic acid sequences [000203] The nucleic acid sequences of the present invention may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, derivatives, mimetics or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns, genie regions, nongenic regions, and regulatory regions. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means. The nucleic acids described herein are used in certain embodiments of the methods of the present invention for production of RNA, proteins or polypeptides, through incorporation into cells, tissues, or organisms. In one embodiment, DNA containing all or part of the coding sequence for the genes described in Tables 2-4, or the SNP markers described in Tables 5-16, is incorporated into a vector for expression of the encoded polypeptide in suitable host cells. The invention also comprises the use of the nucleotide sequence of the nucleic acids of this invention to identify DNA probes for the genes described in Tables 2-4 or the SNP markers described in Tables 5-16, PCR primers to amplify the genes described in Tables 2-4 or the SNP markers described in Tables 5-16, nucleotide polymorphisms in the genes described in Tables 2-4, and regulatory elements of the genes described in Tables 2-4. The nucleic acids of the present invention find use as primers and templates for the recombinant production of ENDOMETRIOSIS disease- associated peptides or polypeptides, for chromosome and gene mapping, to provide antisense sequences, for tissue distribution studies, to locate and obtain full length genes, to identify and obtain homologous sequences (wild-type and mutants), and in diagnostic applications.
Antisense oligonucleotides
[000204] In a particular embodiment of the invention, an antisense nucleic acid or oligonucleotide is wholly or partially complementary to, and can hybridize with, a target nucleic acid (either DNA or RNA) having the sequence of SEQ ID NO:1 , NO:3 or any SEQ ID from any Tables of the invention. For example, an antisense nucleic acid or oligonucleotide comprising 16 nucleotides can be sufficient to inhibit expression of at least one gene from Tables 2-4. Alternatively, an antisense nucleic acid or oligonucleotide can be complementary to 5' or 31 untranslated regions, or can overlap the translation initiation codon (51 untranslated and translated regions) of at least one gene from Tables 2-4, or its functional equivalent. In another embodiment, the antisense nucleic acid is wholly or partially complementary to, and can hybridize with, a target nucleic acid that encodes a polypeptide from a gene described in Tables 2-4.
[000205] In addition, oligonucleotides can be constructed which will bind to duplex nucleic acid (i.e., DNA:DNA or DNA:RNA), to form a stable triple helix containing or triplex nucleic acid. Such triplex oligonucleotides can inhibit transcription and/or expression of a gene from Tables 2-4, or its functional equivalent (M. D. Frank-Kamenetskii et al., 1995). Triplex oligonucleotides are constructed using the basepairing rules of triple helix formation and the nucleotide sequence of the genes described in Tables 2-4.
[000206] The present invention encompasses methods of using oligonucleotides in antisense inhibition of the function of the genes from Tables 2- 4. In the context of this invention, the term "oligonucleotide" refers to naturally- occurring species or synthetic species formed from naturally-occurring subunits or their close homologs. The term may also refer to moieties that function similarly to oligonucleotides, but have non-naturally-occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. In preferred embodiments, at least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure that functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non- chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention. Oligonucleotides may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some non- limiting examples of modifications at the 2' position of sugar moieties which are useful in the present invention include OH, SH, SCH3, F, OCH3, OCN, O(CH2), NH2 and O(CH2)n CH3, where n is from 1 to about 10. Such oligonucleotides are functionally interchangeable with natural oligonucleotides or synthesized oligonucleotides, which have one or more differences from the natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with at least one gene from Tables 2-4 DNA or RNA to inhibit the function thereof.
[000207] The oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits. As defined herein, a "subunit" is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds.
[000208] Antisense nucleic acids or oligonucleotides can be produced by standard techniques (see, e.g., Shewmaker et ai, U.S. Patent No. 6,107,065). The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Any other means for such synthesis may also be employed; however, the actual synthesis of the oligonucleotides is well within the abilities of the practitioner. It is also well known to prepare other oligonucleotides such as phosphorothioates and alkylated derivatives.
[000209] The oligonucleotides of this invention are designed to be hybridizable with RNA (e.g., mRNA) or DNA from genes described in Tables 2-4. For example, an oligonucleotide (e.g., DNA oligonucleotide) that hybridizes to mRNA from a gene described in Tables 2-4 can be used to target the mRNA for RnaseH digestion. Alternatively an oligonucleotide that can hybridize to the translation initiation site of the mRNA of a gene described in Tables 2-4 can be used to prevent translation of the mRNA. In another approach, oligonucleotides that bind to the double-stranded DNA of a gene from Tables 2-4 can be administered. Such oligonucleotides can form a triplex construct and inhibit the transcription of the DNA encoding polypeptides of the genes described in Tables 2-4. Triple helix pairing prevents the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described (see, e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY).
[000210] As non-limiting examples, antisense oligonucleotides may be targeted to hybridize to the following regions: mRNA cap region; translation initiation site; translational termination site; transcription initiation site; transcription termination site; polyadenylation signal; 3' untranslated region; 51 untranslated region; 5' coding region; mid coding region; 3' coding region; DNA replication initiation and elondation sites. Preferably, the complementary oligonucleotide is designed to hybridize to the most unique 5' sequence of a gene described in Tables 2-4, including any of about 15-35 nucleotides spanning the 5' coding sequence. In accordance with the present invention, the antisense oligonucleotide can be synthesized, formulated as a pharmaceutical composition, and administered to a subject. The synthesis and utilization of antisense and triplex oligonucleotides have been previously described (e.g., Simon et al., 1999; Barre et al., 2000; Elez et al., 2000; Sauter et al., 2000). [000211] Alternatively, expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express nucleic acid sequence that is complementary to the nucleic acid sequence encoding a polypeptide from the genes described in Tables 2-4. These techniques are described both in Sambrook et al., 1989 and in Ausubel et al., 1992. For example, expression of at least one gene from Tables 2-4 can be inhibited by transforming a cell or tissue with an expression vector that expresses high levels of untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a nonreplicating vector, and even longer if appropriate replication elements are included in the vector system. Various assays may be used to test the ability of gene-specific antisense oligonucleotides to inhibit the expression of at least one gene from Tables 2-4. For example, mRNA levels of the genes described in Tables 2-4 can be assessed by Northern blot analysis (Sambrook et al., 1989; Ausubel et al., 1992; J. C. Alwine et al. 1977; I. M. Bird, 1998), quantitative or semi-quantitative RT-PCR analysis (see, e.g., W.M. Freeman et al., 1999; Ren et al., 1998; J. M. CaIe et al., 1998), or in situ hybridization (reviewed by A.K. Raap, 1998). Alternatively, antisense oligonucleotides may be assessed by measuring levels of the polypeptide from the genes described in Tables 2-4, e.g., by western blot analysis, indirect immunofluorescence and immunoprecipitation techniques (see, e.g., J. M. Walker, 1998, Protein Protocols on cD-ROM, Humana Press, Totowa, NJ). Any other means for such detection may also be employed, and is well within the abilities of the practitioner.
Mapping Technologies
[000212] The present invention includes various methods which employ mapping technologies to map SNPs and polymorphisms. For purpose of clarity, this section comprises, but is not limited to, the description of mapping technologies that can be utilized to achieve the embodiments described herein. Mapping technologies may be based on amplification methods, restriction enzyme cleavage methods, hybridization methods, sequencing methods, and cleavage methods using agents.
[000213] Amplification methods include: self sustained sequence replication (Guatelli et ai, 1990), transcriptional amplification system (Kwoh et al., 1989), Q- Beta Replicase (Lizardi et al., 1988), isothermal amplification (e.g. Dean et al., 2002; and Hafner et al., 2001), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of ordinary skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low number.
[000214] Restriction enzyme cleavage methods include: isolating sample and control DNA, amplification (optional), digestion with one or more restriction endonucleases, determination of fragment length sizes by gel electrophoresis and comparing samples and controls. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, sequence specific ribozymes (see, e.g., U.S. Pat. No. 5,498,531 or DNAzyme e.g. U.S. Pat. No. 5,807,718) can be used to score for the presence of specific mutations by development or loss of a ribozyme or DNAzyme cleavage site.
[000215] Hybridization methods include any measurement of the hybridization or gene expression levels, of sample nucleic acids to probes corresponding to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, 100, 200, 500, 1000 or more genes, or ranges of these numbers, such as about 5-20, about 10-20, about 20- 50, about 50-100, or about 100-200 genes of Tables 2-4.
[000216] SNPs and SNP maps of the invention can be identified or generated by hybridizing sample nucleic acids, e.g., DNA or RNA, to high density arrays or bead arrays containing oligonucleotide probes corresponding to the polymorphisms of Tables 5-16 (see the Affymetrix arrays and lllumina bead sets at www.affymetrix.com and www.illumina.com and see Cronin et a/., 1996; or Kozal ef a/., 1996).
[000217] Methods of forming high density arrays of oligonucleotides with a minimal number of synthetic steps are known. The oligonucleotide analogue array can be synthesized on a single or on multiple solid substrates by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling (see Pirrung, U.S. Patent No. 5,143,854).
[000218] In brief, the light-directed combinatorial synthesis of oligonucleotide arrays on a glass surface precedes using automated phosphoramidite chemistry and chip masking techniques. In one specific implementation, a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group. Photolysis through a photolithogaphic mask is used selectively to expose functional groups which are then ready to react with incoming 5' photoprotected nucleoside phosphoramidites. The phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group). Thus, the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents.
[000219] In addition to the foregoing, additional methods which can be used to generate an array of oligonucleotides on a single substrate are described in PCT Publication Nos. WO 93/09668 and WO 01/23614. High density nucleic acid arrays can also be fabricated by depositing pre-made or natural nucleic acids in predetermined positions. Synthesized or natural nucleic acids are deposited on specific locations of a substrate by light directed targeting and oligonucleotide directed targeting. Another embodiment uses a dispenser that moves from region to region to deposit nucleic acids in specific spots.
[000220] Nucleic acid hybridization simply involves contacting a probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. See WO 99/32660. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA: DNA, RNA: RNA, or RNA: DNA) will form even where the annealed sequences are not perfectly complementary. Thus, specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization tolerates fewer mismatches. One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency.
[000221] In a preferred embodiment, hybridization is performed at low stringency to ensure hybridization and then subsequent washes are performed at higher stringency to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level control, normalization control, mismatch controls, etc.).
[000222] In general, there is a tradeoff between hybridization specificity (stringency) and signal intensity. Thus, in a preferred embodiment, the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest.
[000223] Probes based on the sequences of the genes described above may be prepared by any commonly available method. Oligonucleotide probes for screening or assaying a tissue or cell sample are preferably of sufficient length to specifically hybridize only to appropriate, complementary genes or transcripts. Typically the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some cases, longer probes of at least 30, 40, or 50 nucleotides will be desirable.
[000224] As used herein, oligonucleotide sequences that are complementary to one or more of the genes or gene fragments described in Tables 2-4 refer to oligonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequences of said genes. Such hybridizable oligonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes (see GeneChip® Expression Analysis Manual, Affymetrix, Rev. 3, which is herein incorporated by reference in its entirety).
[000225] The phrase "hybridizing specifically to" or "specifically hybridizes" refers to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
[000226] As used herein a "probe" is defined as a nucleic acid, capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, U, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
[000227] A variety of sequencing reactions known in the art can be used to directly sequence nucleic acids for the presence or the absence of one or more polymorphisms of Tables 5-16. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) or Sanger (1977). It is also contemplated that any of a variety of automated sequencing procedures can be utilized, including sequencing by mass spectrometry (see, e.g. PCT International Publication No. WO 94/16101 ; Cohen et al., 1996; and Griffin et a/., 1993), real-time pyrophosphate sequencing method (Ronaghi et a/., 1998; and Permutt et al., 2001) and sequencing by hybridization (see e.g. Drmanac et al., 2002).
[000228] Other methods of detecting polymorphisms include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985). In general, the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing a wild-type sequence with potentially mutant RNA or DNA obtained from a sample. The double- stranded duplexes are treated with an agent who cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of a mutation or SNP (see, for example, Cotton et al., 1988; and Saleeba et al., 1992). In a preferred embodiment, the control DNA or RNA can be labeled for detection.
[000229] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping polymorphisms. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches (Hsu et al., 1994). Other examples include, but are not limited to, the MutHLS enzyme complex of E. coli (Smith and Modrich Proc. 1996) and CeI 1 from the celery (Kulinski et al., 2000) both cleave the DNA at various mismatches. According to an exemplary embodiment, a probe based on a polymorphic site corresponding to a polymorphism of Tables 5-16 is hybridized to a cDNA or other DNA product from a test cell or cells. The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039. Alternatively, the screen can be performed in vivo following the insertion of the heteroduplexes in an appropriate vector. The whole procedure is known to those ordinary skilled in the art and is referred to as mismatch repair detection (see e.g. Fakhrai-Rad et al., 2004).
[000230] In other embodiments, alterations in electrophoretic mobility can be used to identify polymorphisms in a sample. For example, single strand conformation polymorphism (SSCP) analysis can be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al., 1989; Cotton et al., 1993; and Hayashi 1992). Single-stranded DNA fragments of case and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence. The resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Kee et al., 1991).
[000231] In yet another embodiment, the movement of mutant or wild-type fragments in a polyacrylamide gel containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al., 1985). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum et al., 1987). In another embodiment, the mutant fragment is detected using denaturing HPLC (see e.g. Hoogendoom et al., 2000).
[000232] Examples of other techniques for detecting polymorphisms include, but are not limited to, selective oligonucleotide hybridization, selective amplification, selective primer extension, selective ligation, single-base extension, selective termination of extension or invasive cleavage assay. For example, oligonucleotide primers may be prepared in which the polymorphism is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al., 1986; Saiki et al., 1989). Such oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, the amplification, the allele-specific hybridization and the detection can be done in a single assay following the principle of the 5' nuclease assay (e.g. see Livak et al., 1995). For example, the associated allele, a particular allele of a polymorphic locus, or the like is amplified by PCR in the presence of both allele-specific oligonucleotides, each specific for one or the other allele. Each probe has a different fluorescent dye at the 5' end and a quencher at the 3' end. During PCR, if one or the other or both allele-specific oligonucleotides are hybridized to the template, the Taq polymerase via its 5' exonuclease activity will release the corresponding dyes. The latter will thus reveal the genotype of the amplified product.
[000233] Hybridization assays may also be carried out with a temperature gradient following the principle of dynamic allele-specific hybridization or like e.g. Jobs et al., (2003); and Bourgeois and Labuda, (2004). For example, the hybridization is done using one of the two allele-specific oligonucleotides labeled with a fluorescent dye, and an intercalating quencher under a gradually increasing temperature. At low temperature, the probe is hybridized to both the mismatched and full-matched template. The probe melts at a lower temperature when hybridized to the template with a mismatch. The release of the probe is captured by an emission of the fluorescent dye, away from the quencher. The probe melts at a higher temperature when hybridized to the template with no mismatch. The temperature-dependent fluorescence signals therefore indicate the absence or presence of an associated allele, a particular allele of a polymorphic locus, or the like (e.g. Jobs et al., 2003). Alternatively, the hybridization is done under a gradually decreasing temperature. In this case, both allele-specific oligonucleotides are hybridized to the template competitively. At high temperature none of the two probes are hybridized. Once the optimal temperature of the full-matched probe is reached, it hybridizes and leaves no target for the mismatched probe (e.g. Bourgeois and Labuda, 2004). In the latter case, if the allele-specific probes are differently labeled, then they are hybridized to a single PCR-amplified target. If the probes are labeled with the same dye, then the probe cocktail is hybridized twice to identical templates with only one labeled probe, different in the two cocktails, in the presence of the unlabeled competitive probe.
[000234] Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification may carry the associated allele, a particular allele of a polymorphic locus, or the like, also referred to as "mutation" of interest in the center of the molecule, so that amplification depends on differential hybridization (Gibbs et al., 1989) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, 1993). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., 1992). It is anticipated that in certain embodiments, amplification may also be performed using Taq ligase for amplification (Barany, 1991). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known associated allele, a particular allele of a polymorphic locus, or the like at a specific site by looking for the presence or absence of amplification. The products of such an oligonucleotide ligation assay can also be detected by means of gel electrophoresis. Furthermore, the oligonucleotides may contain universal tags used in PCR amplification and zip code tags that are different for each allele. The zip code tags are used to isolate a specific, labeled oligonucleotide that may contain a mobility modifier (e.g. Grossman et al., 1994).
[000235] In yet another alternative, allele-specific elongation followed by ligation will form a template for PCR amplification. In such cases, elongation will occur only if there is a perfect match at the 3' end of the allele-specific oligonucleotide using a DNA polymerase. This reaction is performed directly on the genomic DNA and the extension/ligation products are amplified by PCR. To this end, the oligonucleotides contain universal tags allowing amplification at a high multiplex level and a zip code for SNP identification. The PCR tags are designed in such a way that the two alleles of a SNP are amplified by different forward primers, each having a different dye. The zip code tags are the same for both alleles of a given SNPs and they are used for hybridization of the PCR- amplified products to oligonucleotides bound to a solid support, chip, bead array or like. For an example of the procedure, see Fan et al. (Cold Spring Harbor Symposia on Quantitative Biology, Vol. LXVIII, pp. 69-78 2003).
[000236] Another alternative includes the single-base extension/ligation assay using a molecular inversion probe, consisting of a single, long oligonucleotide (see e.g. Hardenbol et al., 2003). In such an embodiment, the oligonucleotide hybridizes on both side of the SNP locus directly on the genomic DNA, leaving a one-base gap at the SNP locus. The gap-filling, one-base extension/ligation is performed in four tubes, each having a different dNTP. Following this reaction, the oligonucleotide is circularized whereas unreactive, linear oligonucleotides are degraded using an exonuclease such as exonuclease I of E. coli. The circular oligonucleotides are then linearized and the products are amplified and labeled using universal tags on the oligonucleotides. The original oligonucleotide also contains a SNP-specific zip code allowing hybridization to oligonucleotides bound to a solid support, chip, and bead array or like. This reaction can be performed at a high multiplexed level.
[000237] In another alternative, the associated allele, a particular allele of a polymorphic locus, or the like is scored by single-base extension (see e.g. U.S. Pat. No. 5,888,819). The template is first amplified by PCR. The extension oligonucleotide is then hybridized next to the SNP locus and the extension reaction is performed using a thermostable polymerase such as ThermoSequenase (GE Healthcare) in the presence of labeled ddNTPs. This reaction can therefore be cycled several times. The identity of the labeled ddNTP incorporated will reveal the genotype at the SNP locus. The labeled products can be detected by means of gel electrophoresis, fluorescence polarization (e.g. Chen et al., 1999) or by hybridization to oligonucleotides bound to a solid support, chip, and bead array or like. In the latter case, the extension oligonucleotide will contain a SNP-specific zip code tag.
[000238] In yet another alternative, a SNP is scored by selective termination of extension. The template is first amplified by PCR and the extension oligonucleotide hybridizes in the vicinity of the SNP locus, close to but not necessarily adjacent to it. The extension reaction is carried out using a thermostable polymerase such as ThermoSequenase (GE Healthcare) in the presence of a mix of dNTPs and at least one ddNTP. The latter has to terminate the extension at one of the allele of the interrogated SNP, but not both such that the two alleles will generate extension products of different sizes. The extension product can then be detected by means of gel electrophoresis, in which case the extension products need to be labeled, or by mass spectrometry (see e.g. Storm et al., 2003).
[000239] In another alternative, SNPs are detected using an invasive cleavage assay (see U.S. Pat. No. 6,090,543). There are five oligonucleotides per SNP to interrogate but these are used in a two step-reaction. During the primary reaction, three of the designed oligonucleotides are first hybridized directly to the genomic DNA. One of them is locus-specific and hybridizes up to the SNP locus (the pairing of the 3' base at the SNP locus is not necessary). There are two allele- specific oligonucleotides that hybridize in tandem to the locus-specific probe but also contain a 5' flap that is specific for each allele of the SNP. Depending upon hybridization of the allele-specific oligonucleotides at the base of the SNP locus, this creates a structure that is recognized by a cleavase enzyme (U.S. Pat. No. 6,090,606) and the allele-specific flap is released. During the secondary reaction, the flap fragments hybridize to a specific cassette to recreate the same structure as above except that the cleavage will release a small DNA fragment labeled with a fluorescent dye that can be detected using regular fluorescence detector. In the cassette, the emission of the dye is inhibited by a quencher.
Methods to identify agents that modulate the expression of a nucleic acid encoding a gene involved in ENDOMETRIOSIS
[000240] The present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a gene from Tables 2-4. Such methods may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down- regulating expression of the nucleic acid in a cell. Such cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium. Some non-limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
[000241] In one assay format, the expression of a nucleic acid encoding a gene of the invention (see Tables 2-4) in a cell or tissue sample is monitored directly by hybridization to the nucleic acids of the invention. Cell lines or tissues are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such as those disclosed in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).
[000242] Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared as described above. Hybridization conditions are modified using known methods, such as those described by Sambrook et al., and Ausubel et al., as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip or a porous glass wafer. The chip or wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize to the RNA. By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate expression are identified.
Methods to identify agents that modulate the activity of a protein encoded by a gene involved in ENDOMETRIOSIS and antibodies of the invention [000243] The present invention provides methods for identifying agents that modulate at least one activity of the proteins described in Tables 2-4. Such methods may utilize any means of monitoring or detecting the desired activity. As used herein, an agent is said to modulate the expression of a protein of the invention if it is capable of up- or down- regulating expression of the protein in a cell. Such cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium. Some non- limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
[000244] In one format, the specific activity of a protein of the invention, normalized to a standard unit, may be assayed in a cell population that has been exposed to the agent to be tested and compared to an unexposed control cell population. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and times. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with a probe, such as an antibody probe.
[000245] Antibodies and Antibody probes can be prepared by immunizing suitable mammalian (e.g. mice or transgenic mice) hosts utilizing appropriate immunization protocols using the proteins of the invention or antigen-containing fragments thereof. To enhance immunogenicity, these proteins or fragments can be conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL) may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using standard methods, see e.g., Kohler & Milstein (1992) or modifications which affect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies can be screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid. The desired monoclonal antibodies may be recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant portion(s) can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as Fab or Fab' fragments, is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin. The antibody chains (light and heavy) can be cloned into the vector by methods known in the art. The antibodies or fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras derived from multiple species. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras from multiple species, for instance, humanized antibodies. The antibody can therefore be a humanized antibody or a human antibody, as described in U.S. Patent 5,585,089 or Riechmann et a/. (1988).
[000246] Phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for proteins, or fragments thereof, described in Tables 2-4. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al., EMBO J., 13:3245- 3260 (1994); Nissim et al., ibid, pp. 692-698 and by Griffiths et al., ibid, 12:725- 734. The antibody of the invention also comprise humanized and human antibodies. Such antibodies are made by methods known in the art.
[000247] Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a non- random basis which takes into account the sequence of the target site or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site. The agents of the present invention can be, as examples, oligonucleotides, antisense polynucleotides, interfering RNA, peptides, peptide mimetics, antibodies, antibody fragments, small molecules, vitamin derivatives, as well as carbohydrates. Peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene- encoded amino acids are to be included.
[000248] Another class of agents of the present invention includes antibodies or fragments thereof that bind to a protein encoded by a gene in Tables 2-4. Antibody agents can be obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies (see section above of antibodies as probes for standard antibody preparation methodologies).
[000249] In yet another class of agents, the present invention includes peptide mimetics that mimic the three-dimensional structure of the protein encoded by a gene from Tables 2-4. Such peptide mimetics may have significant advantages over naturally occurring peptides, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half- life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity and others. In one form, mimetics are peptide-containing molecules that mimic elements of protein secondary structure. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. In another form, peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are also referred to as peptide mimetics or peptidomimetics (Fauchere, 1986; Veber & Freidinger, 1985; Evans et a/., 1987) which are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptide mimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage using methods known in the art. Labeling of peptide mimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptide mimetic that are predicted by quantitative structure-activity data and molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macromolecule(s) to which the peptide mimetic binds to produce the therapeutic effect. Derivitization (e.g., labeling) of peptide mimetics should not substantially interfere with the desired biological or pharmacological activity of the peptide mimetic. The use of peptide mimetics can be enhanced through the use of combinatorial chemistry to create drug libraries. The design of peptide mimetics can be aided by identifying amino acid mutations that increase or decrease binding of the protein to its binding partners. Approaches that can be used include the yeast two hybrid method (see Chien et al., 1991) and the phage display method. The two hybrid method detects protein- protein interactions in yeast (Fields et al., 1989). The phage display method detects the interaction between an immobilized protein and a protein that is expressed on the surface of phages such as lambda and M 13 (Amberg et al., 1993; Hogrefe et al., 1993). These methods allow positive and negative selection for protein-protein interactions and the identification of the sequences that determine these interactions.
Method to diagnose ENDOMETRIOSIS
[000250] The present invention also relates to methods for diagnosing ENDOMETRIOSIS or a related disease, preferably a subtype of ENDOMETRIOSIS, a predisposition to such a disease and/or disease progression. In some methods, the steps comprise contacting a target sample with (a) nucleic acid molecule(s) or fragments thereof and comparing the concentration of individual mRNA(s) with the concentration of the corresponding mRNA(s) from at least one healthy donor. An aberrant (increased or decreased) mRNA level of at least one gene from Tables 2-4, at least 5 or 10 genes from Tables 2-4, at least 50 genes from Tables 2-4, at least 100 genes from Tables 2- 4 or at least 200 genes from Tables 2-4 determined in the sample in comparison to the control sample is an indication of ENDOMETRIOSIS disease or a related subtype or a disposition to such kinds of diseases. For diagnosis, samples are, preferably, obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium. Some non-limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells.
[000251] For analysis of gene expression, total RNA is obtained from cells according to standard procedures and, preferably, reverse-transcribed. Preferably, a DNAse treatment (in order to get rid of contaminating genomic DNA) is performed.
[000252] The nucleic acid molecule or fragment is typically a nucleic acid probe for hybridization or a primer for PCR. The person skilled in the art is in a position to design suitable nucleic acids probes based on the information provided in the Tables of the present invention. The target cellular component, i.e. mRNA, e.g., in brain tissue, may be detected directly in situ, e.g. by in situ hybridization or it may be isolated from other cell components by common methods known to those skilled in the art before contacting with a probe. Detection methods include Northern blot analysis, RNase protection, in situ methods, e.g. in situ hybridization, in vitro amplification methods (PCR, LCR, QRNA replicase or RNA-transcription/amplification (TAS, 3SR), reverse dot blot disclosed in EP-B10237362) and other detection assays that are known to those skilled in the art. Products obtained by in vitro amplification can be detected according to established methods, e.g. by separating the products on agarose or polyacrylamide gels and by subsequent staining with ethidium bromide or any other dye or reagent. Alternatively, the amplified products can be detected by using labeled primers for amplification or labeled dNTPs. Preferably, detection is based on a microarray.
[000253] The probes (or primers) (or, alternatively, the reverse-transcribed sample mRNAs) can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme. [000254] The present invention also relates to the use of the nucleic acid molecules or fragments described above for the preparation of a diagnostic composition for the diagnosis of ENDOMETRIOSIS or a subtype or predisposition to such a disease.
[000255] The present invention also relates to the use of the nucleic acid molecules of the present invention for the isolation or development of a compound which is useful for therapy of ENDOMETRIOSIS. For example, the nucleic acid molecules of the invention and the data obtained using said nucleic acid molecules for diagnosis of ENDOMETRIOSIS might allow for the identification of further genes which are specifically dysregulated, and thus may be considered as potential targets for therapeutic interventions. Furthermore, such diagnostic might also be used for selection of patients that might respond positively or negatively to a potential target for therapeutic interventions (as for the pharmacogenomics and personalized medicine concept well know in the art; see prognostic assays text below).
[000256] The invention further provides prognostic assays that can be used to identify subjects having or at risk of developing ENDOMETRIOSIS. In such method, a test sample is obtained from a subject and the amount and/or concentration of the nucleic acid described in Tables 2-4 is determined; wherein the presence of an associated allele, a particular allele of a polymorphic locus, or the likes in the nucleic acids sequences of this invention (see SEQ ID from Tables 5-16) can be diagnostic for a subject having or at risk of developing ENDOMETRIOSIS. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid, a cell sample, or tissue. A biological fluid can be, but is not limited to saliva, serum, mucus, urine, stools, spermatozoids, vaginal secretions, lymph, amiotic liquid, pleural liquid and tears. Cells can be, but are not limited to: ovarian cells, uterus cells and other cells of the reproductive system, hair cells, muscle cells, nervous cells, blood and vessels cells, dermis, epidermis and other skin cells, and various brain cells. [000257] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic acid such as antisense DNA or interfering RNA (RNAi), small molecule or other drug candidate) to treat ENDOMETRIOSIS. Specifically, these assays can be used to predict whether an individual will have an efficacious response or will experience adverse events in response to such an agent. For example, such methods can be used to determine whether a subject can be effectively treated with an agent that modulates the expression and/or activity of a gene from Tables 2-4 or the nucleic acids described herein. In another example, an association study may be performed to identify polymorphisms from Tables 5-16 that are associated with a given response to the agent, e.g., an efficacious response or the likelihood of one or more adverse events. Thus, one embodiment of the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant expression or activity of a gene from Tables 2-4 in which a test sample is obtained and nucleic acids or polypeptides from Tables 2-4 are detected (e.g., wherein the presence of a particular level of expression of a gene from Tables 2-4 or a particular allelic variant of such gene, such as polymorphisms from Tables 5-16 is diagnostic for a subject that can be administered an agent to treat a disorder such as ENDOMETRIOSIS). In one embodiment, the method includes obtaining a sample from a subject suspected of having ENDOMETRIOSIS or an affected individual and exposing such sample to an agent. The expression and/or activity of the nucleic acids and/or genes of the invention are monitored before and after treatment with such agent to assess the effect of such agent. After analysis of the expression values, one skilled in the art can determine whether such agent can effectively treat such subject. In another embodiment, the method includes obtaining a sample from a subject having or susceptible to developing ENDOMETRIOSIS and determining the allelic constitution of polymorphisms from Tables 5-16 that are associated with a particular response to an agent. After analysis of the allelic constitution of the individual at the associated polymorphisms, one skilled in the art can determine whether such agent can effectively treat such subject. [000258] The methods of the invention can also be used to detect genetic alterations in a gene from Tables 2-4, thereby determining if a subject with the lesioned gene is at risk for a disease associated with ENDOMETRIOSIS. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration linked to or affecting the integrity of a gene from Tables 2-4 encoding a polypeptide or the misexpression of such gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of: (1) a deletion of one or more nucleotides from a gene from Tables 2-4; (2) an addition of one or more nucleotides to a gene from Tables 2-4; (3) a substitution of one or more nucleotides of a gene from Tables 2-4; (4) a chromosomal rearrangement of a gene from Tables 2-4; (5) an alteration in the level of a messenger RNA transcript of a gene from Tables 2-4; (6) aberrant modification of a gene from Tables 2-4, such as of the methylation pattern of the genomic DNA, (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a gene from Tables 2-4; (8) inappropriate post-translational modification of a polypeptide encoded by a gene from Tables 2-4; and (9) alternative promoter use. As described herein, there are a large number of assay techniques known in the art which can be used for detecting alterations in a gene from Tables 2-4. A preferred biological sample is a peripheral blood sample obtained by conventional means from a subject. Another preferred biological sample is a buccal swab. Other biological samples can be, but are not limited to, urine, stools, vaginal secretions, lymph, amiotic liquid, pleural liquid and tears.
[000259] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et a/., 1988; and Nakazawa et ai, 1994), the latter of which can be particularly useful for detecting point mutations in a gene from Tables 2-4 (see Abavaya et a/., 1995). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic DNA, mRNA, or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene from Tables 2-4 under conditions such that hybridization and amplification of the nucleic acid from Tables 2-4 (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with some of the techniques used for detecting a mutation, an associated allele, a particular allele of a polymorphic locus, or the like described in the above sections. Other mutation detection and mapping methods are described in previous sections of the detailed description of the present invention.
[000260] The present invention also relates to further methods for diagnosing ENDOMETRIOSIS or a related disorder or subtype, a predisposition to such a disorder and/or disorder progression. In some methods, the steps comprise contacting a target sample with (a) nucleic molecule(s) or fragments thereof and determining the presence or absence of a particular allele of a polymorphism that confers a disorder-related phenotype (e.g., predisposition to such a disorder and/or disorder progression). The presence of at least one allele from Tables 5- 16 that is associated with ENDOMETRIOSIS ("associated allele"), at least 5 or 10 associated alleles from Tables 5-16, at least 50 associated alleles from Tables 5- 16 at least 100 associated alleles from Tables 5-16, or at least 200 associated alleles from Tables 5-16 determined in the sample is an indication of ENDOMETRIOSIS disease or a related disorder, a disposition or predisposition to such kinds of disorders, or a prognosis for such disorder progression. Such samples and cells can be obtained from any parts of the body such as the hair, mouth, rectum, scalp, blood, dermis, epidermis, skin cells, cutaneous surfaces, intertrigious areas, genitalia and fluids, vessels and endothelium. Some non- limiting examples of cells that can be used are: ovarian cells, uterus cells and other cells of the reproductive system, muscle cells, nervous cells, blood and vessels cells, T cell, mast cell, lymphocyte, monocyte, macrophage, and epithelial cells. [000261] In other embodiments, alterations in a gene from Tables 2-4 can be identified by hybridizing sample and control nucleic acids, e.g., DNA or RNA, to high density arrays or bead arrays containing tens to thousands of oligonucleotide probes (Cronin et al., 1996; Kozal et al., 1996). For example, alterations in a gene from Tables 2-4 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al., (1996). Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations, associated alleles, particular alleles of a polymorphic locus, or the like. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants, mutations, alleles detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
[000262] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence a gene from Tables 2-4 and detect an associated allele, a particular allele of a polymorphic locus, or the like by comparing the sequence of the sample gene from Tables 2-4 with the corresponding wild-type (control) sequence (see text described in previous sections for various sequencing techniques and other methods of detecting an associated allele, a particular allele of a polymorphic locus, or the likes in a gene from Tables 2-4. Such methods include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA, DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985) and alterations in electrophoretic mobility. Examples of other techniques for detecting point mutations, an associated allele, a particular allele of a polymorphic locus, or the like include, but are not limited to, selective oligonucleotide hybridization, selective amplification, selective primer extension, selective ligation, single-base extension, selective termination of extension or invasive cleavage assay.
[000263] Other types of markers can also be used for diagnostic purposes. For example, microsatellites can also be useful to detect the genetic predisposition of an individual to a given disorder. Microsatellites consist of short sequence motifs of one or a few nucleotides repeated in tandem. The most common motifs are polynucleotide runs, dinucleotide repeats (particularly the CA repeats) and trinucleotide repeats. However, other types of repeats can also be used. The microsatellites are very useful for genetic mapping because they are highly polymorphic in their length. Microsatellite markers can be typed by various means, including but not limited to DNA fragment sizing, oligonucleotide ligation assay and mass spectrometry. For example, the locus of the microsatellite is amplified by PCR and the size of the PCR fragment will be directly correlated to the length of the microsatellite repeat. The size of the PCR fragment can be detected by regular means of gel electrophoresis. The fragment can be labeled internally during PCR or by using end-labeled oligonucleotides in the PCR reaction (e.g. Mansfield et a/., 1996). Alternatively, the size of the PCR fragment is determined by mass spectrometry. In another alternative, an oligonucleotide ligation assay can be performed. The microsatellite locus is first amplified by PCR. Then, different oligonucleotides can be submitted to ligation at the center of the repeat with a set of oligonucleotides covering all the possible lengths of the marker at a given locus (Zirvi et ai, 1999). Another example of design of an oligonucleotide assay comprises the ligation of three oligonucleotides; a 5' oligonucleotide hybridizing to the 5' flanking sequence, a repeat oligonucleotide of the length of the shortest allele of the marker hybridizing to the repeated region and a set of 3' oligonucleotides covering all the existing alleles hybridizing to the 3' flanking sequence and a portion of the repeated region for all the alleles longer than the shortest one. For the shortest allele, the 3' oligonucleotide exclusively hybridizes to the 3' flanking sequence (U.S. Pat. No. 6,479,244).
[000264] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid selected from the SEQ ID of Tables 5-16, or antibody reagent described herein, which may be conveniently used, for example, in a clinical setting to diagnose patient exhibiting symptoms or a family history of a disorder or disorder involving abnormal activity of genes from Tables 2-4.
Method to treat an animal suspected of having ENDOMETRIOSIS
[000265] The present invention provides methods of treating a disease associated with ENDOMETRIOSIS disease by expressing in vivo the nucleic acids of at least one gene from Tables 2-4. These nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms as described below. The nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell. The nucleic acids encoding a gene from Tables 2-4, under the control of a promoter, then express the encoded protein, thereby mitigating the effects of absent, partial inactivation, or abnormal expression of a gene from Tables 2-4.
[000266] Such gene therapy procedures have been used to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts. The ability to express artificial genes in humans facilitates the prevention and/or cure of many important human disorders, including many disorders which are not amenable to treatment by other therapies (for a review of gene therapy procedures, see Anderson, 1992; Nabel & Feigner, 1993; Mitani & Caskey, 1993; Mulligan, 1993; Dillon, 1993; Miller, 1992; Van Brunt, 1998; Vigne, 1995; Kremer & Perricaudet 1995; Doerfler & Bohm 1995; and Yu et al., 1994).
[000267] Delivery of the gene or genetic material into the cell is the first critical step in gene therapy treatment of a disorder. A large number of delivery methods are well known to those of skill in the art. Preferably, the nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see the references included in the above section.
[000268] The use of RNA or DNA based viral systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[000269] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non- dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of c/s-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian lmmuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., 1992; Johann et al., 1992; Sommerfelt et al., 1990; Wilson et a/., 1989; Miller et al., 1999;and PCT/US94/05700). [000270] In applications where transient expression of the nucleic acid is preferred, adenoviral based systems are typically used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., 1987; U.S. Pat. No. 4,797,368; WO 93/24641 ; Kotin, 1994; Muzyczka, 1994). Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., 1985; Tratschin, et al., 1984; Hermonat & Muzyczka, 1984; and Samulski et al., 1989.
[000271] In particular, numerous viral vector approaches are currently available for gene transfer in clinical trials, with retroviral vectors by far the most frequently used system. All of these viral vectors utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials (Dunbar et al., 1995; Kohn et al., 1995; Malech et al., 1997). PA317/pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al., 1995). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors (Ellem et al., 1997; and Dranoff et al., 1997).
[000272] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system (Wagner et al., 1998, Kearns et al., 1996). [000273] Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used in transient expression gene therapy; because they can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply the deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle tissues. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., 1998). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., 1996; Sterman et al., 1998; Welsh et al., 1995; Alvarez et al., 1997; Topf et al., 1998.
[000274] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
[000275] In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. A viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., 1995, reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other pairs of viruses expressing a ligand fusion protein and target cells expressing a receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., Fab or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.
[000276] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, and tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
[000277] Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et a/., 1994; and the references cited therein for a discussion of how to isolate and culture cells from patients).
[000278] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known (see Inaba ef a/., 1992).
[000279] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells).
[000280] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo. Alternatively, naked DNA can be administered.
[000281] Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells, as described above. The nucleic acids from Tables 2-4 are administered in any suitable manner, preferably with the pharmaceutically acceptable carriers described above. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route (see Samulski et a/., 1989). The present invention is not limited to any method of administering such nucleic acids, but preferentially uses the methods described herein. [000282] The present invention further provides other methods of treating ENDOMETRIOSIS disease such as administering to an individual having ENDOMETRIOSIS disease an effective amount of an agent that regulates the expression, activity or physical state of at least one gene from Tables 2-4. An "effective amount" of an agent is an amount that modulates a level of expression or activity of a gene from Tables 2-4, in a cell in the individual at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more, compared to a level of the respective gene from Tables 2-4 in a cell in the individual in the absence of the compound. The preventive or therapeutic agents of the present invention may be administered, either orally or parenterally, systemically or locally. For example, intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppositories, intestinal lavage, oral enteric coated tablets, and the like can be selected, and the method of administration may be chosen, as appropriate, depending on the age and the conditions of the patient. The effective dosage is chosen from the range of 0.01 mg to 100 mg per kg of body weight per administration. Alternatively, the dosage in the range of 1 to 1000 mg, preferably 5 to 50 mg per patient may be chosen. The therapeutic efficacy of the treatment may be monitored by observing various parts of the reproductive system and other body parts, or any other monitoring methods known in the art. Other ways of monitoring efficacy can be, but are not limited to monitoring pelvic pain, fertility, ovarian cysts formation and progression, or any other ENDOMETRIOSIS related symptom.
[000283] The present invention further provides a method of treating an individual clinically diagnosed with ENDOMETRIOSES' disease. The methods generally comprises analyzing a biological sample that includes a cell, in some cases, a cell, from an individual clinically diagnosed with ENDOMETRIOSIS disease for the presence of modified levels of expression of at least 1 gene, at least 10 genes, at least 50 genes, at least 100 genes, or at least 200 genes from Tables 2-4. A treatment plan that is most effective for individuals clinically diagnosed as having a condition associated with ENDOMETRIOSIS disease is then selected on the basis of the detected expression of such genes in a cell. Treatment may include administering a composition that includes an agent that modulates the expression or activity of a protein from Tables 2-4 in the cell. Information obtained as described in the methods above can also be used to predict the response of the individual to a particular agent. Thus, the invention further provides a method for predicting a patient's likelihood to respond to a drug treatment for a condition associated with ENDOMETRIOSIS disease, comprising determining whether modified levels of a gene from Tables 2-4 is present in a cell, wherein the presence of protein is predictive of the patient's likelihood to respond to a drug treatment for the condition. Examples of the prevention or improvement of symptoms accompanied by ENDOMETRIOSIS disease that can monitored for effectiveness include prevention or improvement of pelvic pain, infertility, or any other ENDOMETRIOSIS related symptom.
[000284] The invention also provides a method of predicting a response to therapy in a subject having ENDOMETRIOSIS disease by determining the presence or absence in the subject of one or more markers associated with ENDOMETRIOSIS disease described in Tables 5-16, diagnosing the subject in which the one or more markers are present as having ENDOMETRIOSIS disease, and predicting a response to a therapy based on the diagnosis e.g., response to therapy may include an efficacious response and/or one or more adverse events. The invention also provides a method of optimizing therapy in a subject having ENDOMETRIOSIS disease by determining the presence or absence in the subject of one or more markers associated with a clinical subtype of ENDOMETRIOSIS disease, diagnosing the subject in which the one or more markers are present as having a particular clinical subtype of ENDOMETRIOSIS disease, and treating the subject having a particular clinical subtype of ENDOMETRIOSIS disease based on the diagnosis. As an example, treatment for the pelvic pain or infertility subtypes of ENDOMETRIOSIS.
[000285] Thus, while there are a number of available treatments to relieve the symptoms of ENDOMETRIOSIS, they all are accompanied by various side effects, high costs, and long complicated treatment protocols, which are often not available and effective in a large number of individuals. Symptoms also often come back shortly after treatments are stopped. Accordingly, there remains a need in the art for more effective and otherwise improved methods for diagnosing, treating and preventing ENDOMETRIOSIS. Thus, there is a continuing need in the medical arts for genetic markers of ENDOMETRIOSIS disease and guidance for the use of such markers. The present invention fulfills this need and provides further related advantages.
EXAMPLES
Example 1 : Identification of cases and controls
[000286] All individuals were sampled from the Quebec founder population (QFP). Membership in the founder population was defined as having four grandparents of the affected child having French Canadian family names and being born in the Province of Quebec, Canada or in adjacent areas of the Provinces of New Brunswick and Ontario or in New England or New York State. The Quebec founder population is expected to have two distinct advantages over general populations for LD mapping: 1) increased LD resulting from a limited number of generations since the founding of the population and 2) increased genetic alleic homogeneity because of the restricted number of founders (estited 2600 effective founders, Charbonneau et al. 1987). Reduced allelic heterogeneity will act to increase relative risk imparted by the remaining alleles and so increase the power of case/control studies to detect genes and gene alleles involved in complex disorders within the Quebec population. The specific combination of age in generations, optimal number of founders and large present population size makes the QFP optimal for LD-based gene mapping.
[000287] All enrolled QFP subjects (patients and controls) provided a 20 ml blood sample (2 barcoded tubes of 10 ml). Samples were processed immediately upon arrival at the laboratory. All samples were scanned and logged into a LabVantage Laboratory Information Management System (LIMS), which served as a hub between the clinical data management system and the genetic analysis system. Following centrifugation, the buffy coat containing the white blood cells was isolated from each tube. Genomic DNA was extracted from the buffy coat from one of the tubes, and stored at 4°C until required for genotyping. DNA extraction was performed with a commercial kit using a guanidine hydrochloride based method (FlexiGene, Qiagen) according to the manufacturer's instructions. The extraction method yielded high molecular weight DNA, and the quality of every DNA sample was verified by agarose gel electrophoresis. Genomic DNA appeared on the gel as a large band of very high molecular weight. The remaining two buffy coats were stored at -800C as backups.
[000288] The QFP samples were collected as cases and controls consisting of ENDOMETRIOSIS disease subjects and controls. 511 cases and 511 controls were used for the analysis reported here.
[000289] The cases had a diagnosis of ENDOMETRIOSIS confirmed by the typical black lesions observed during surgery or laparoscopy, or confirmed by a pathology report. The controls were minimally phenotyped and met the following criteria:
- Female
- 40 years of age or older
- Must have at least one child
- No self-declared ENDOMETRIOSIS
- No other self-declared uterine disease
Example 2: Genome Wide Association
[000290] Genotyping was performed using the QLDM-Max SNP map using lllumina's Infinium-ll technology Single Sample Beadchips. The QLDM-Max map contains 374,187 SNPs. The SNPs are contained in the lllumina HumanHap-300 arrays plus two custom SNP sets of approximately 30,000 markers each. The HumanHap-300 chip includes 317,503 tag SNPs derived from the Phase I HapMap data. The additional (approx.) 60,000 SNPs were selected by to optimize the density of the marker map across the genome matching the LD pattern in the Quebec Founder Population, as established from previous studies at Genizon, and to fill gaps in the lllumina HumanHap-300 map. The SNPs were genotyped on the 459 trios for a total of ~515,255,499 genotypes.
[000291] The genotyping information was entered into a Unified Genotype Database (a proprietary database under development) from which it was accessed using custom-built programs for export to the genetic analysis pipeline. Analyses of these genotypes were performed with the statistical tools described in Example 3. The GWS and the different analyses permitted the identification of candidate chromosomal regions linked to ENDOMETRIOSIS disease (Table 1).
[000292] Example 3: Genetic Analysis
1. Dataset quality assessment
[000293] Prior to performing any analysis, the sample was examined to ascertain that no subjects were related more closely than 5 meiotic steps.
[000294] The data were then subjected to a cleaning step. The program, DataStats was used to calculate the following statistics per marker or per <individual>:
■ Minor allele frequency (MAF) for each marker
Number of markers with MAF < 5%, < 4%,< 3%,< 2%,< 1%
■ Number of missing values for each marker and individual
■ Monomorphic markers
■ Departure from Hardy-Weinberg equilibrium within control individuals for each marker
The following acceptance criteria were required for further analysis:
■ Missing values per marker or individual < 1 %
■ Minor allele frequency per marker > 4 %,
■ Allele frequencies for controls in Hardy-Weinberg equilibrium
Markers and individuals not meeting criteria were removed from the dataset using DataPullPC. If a case or a control was removed by the cleaning process, its region and gender matched case or control were also be removed from the analysis.
2. Phase Determination [000295] Haplotypes will were estimated from the case/control genotype data using ggplem a modified version of the PL-EM algorithm. The programs Qeno2patctr and tapper determined case and control genotypes and prepared the data in the input format for PL-EM. An EM. algorithm module consisting of several applications was used to resolve phase ambiguities. PLEMPre first recoded the genotypes for input into the PL-EM algorithm which used an 11- marker sliding block for haplotype estimation and deposited the constructed haplotypes into a file, happatctr which was the input file for haplotype association analysis performed by the program, LDSTATS.
[000296] The program GeneWriter was used to create a case-control genotype file, penopatctr, which was the input for the program, SiNGLETYPE, which was used to perform single marker case-control association analysis.
3. Haplotype association analysis
[000297] Haplotype association analysis was performed using the program LDSTATS. LDSTATS tests for association of haplotypes with the disease phenotype. The algorithms LDSTATS (v2.0) and LDSTATS (v4.0) define haplotypes using multi-marker windows that advance across the marker map in one-marker increments. Windows of size 1 , 3, 5, 7, and 9 were analyzed. At each position the frequency of haplotypes in cases and controls was determined and a chi-square statistic was calculated from case control frequency tables. For LDSTATS v2.0, the significance of the chi-square for single marker and 3-marker windows was calculated as Pearson's chi-square with degrees of freedom. Larger windows of multi-allelic haplotype association were tested using Smith's normalization of the square root of Pearson's Chi-square.
[000298] LDSTATS v4.0 calculates significance of chi-square values using a permutation test in which case-control status is randomly permuted until 350 permuted chi-square values are observed that are greater than or equal to chi- square value of the actual data. The P value is then calculated as 350 / the number of permutations required. [000299] Tables 5.1-16.1 lists the results for association analysis using LDSTATs (v2.0 and v4.0) for the candidate regions described in Table 1 based on the genome wide scan genotype data for various subphenotypes from the QFP cases and controls. For each one of these regions, we report in Tables 5.2- 16.2 the allele frequencies and the relative risk (RR) for the haplotypes contributing to the best signal at each SNP in the region.
4. Singletype analysis
[000300] The program SINGLETYPE was used to calculate both allelic and genotype association for each single marker, one at a time using the genotype data in the file, genopatctr as input. Allelic association was tested using a 2 X 2 contingency table comparing allele 1 in cases and controls and allele 2 in cases and controls and genotype association was tested using a 2 X 3 contingency table comparing genotype 11 in cases and controls, genotype 12 in cases and controls and genotype 22 in cases and controls. SINGLETYPE was also used to test dominant and recessive models (11 and 12 genotypes combined vs. 22; or 22 and 12 genotypes combined vs. 11).
5. Conditional analysis
[000301] Conditional analyses were performed on subsets of the original set of 511 cases using the program LDSTATS (v2.0). The selection of a subset of cases and their matched controls was based on the carrier status of cases at a gene or locus of interest. We selected genes PRKCE on chromosome 2, RAF1 on chromosome 3, DNAH5 on chromosome 5 and SYNE1 on chromosome 6, based on our association findings using LDSTAT (v2.0). The most significant single SNP association signal in PRKCE, using build 36, was obtained with a SNP corresponding to SEQ ID 4519 (see Table below for conversion to the specific DNA genotypes used). We selected a set of risk genotypes for conditional analyses. The set consisted of genotypes 1/2 and 2/2. Using this set, we partitioned the cases into two groups; the first group consisting of those cases that were carrier of a risk genotype and the second group consisting of the remaining cases, the non-carriers. The resulting sample sizes were respectively 329 and 181. LDSTAT (v2.0) was run in each group and regions showing association with endometriosis are reported in Table 12.1. Regions associated with endometriosis in the group of carriers (has_PRKCE-1_cr) indicate the presence of an epistatic interaction between risk factors in those regions and risk factors in PRKCE (Table 12.2).
[000302] A second conditional analysis was performed using gene RAF1 on chromosome 3. The most significant association in RAF1 , using build 36, was obtained with a SNP corresponding to SEQ ID 4676 (see Table below for conversion to the specific DNA genotypes used). We selected a set of risk genotypes for conditional analyses. The set consisted of genotypes 1/2 and 2/2. Using this risk set, we partitioned the cases into two groups; the first group consisting of those cases that were carrier of a risk genotype and the second group consisting of the remaining cases, the non-carriers. The resulting sample sizes were respectively 222 and 289. LDSTAT (v2.0) was run in each group and regions showing association with endometriosis are reported in Table 13.1. Regions associated with endometriosis in the group of non-carriers (not_RAF1- 1_cr) indicate the existence of risk factors acting independently of RAF 1 (Table 13.2).
[000303] A third conditional analysis was performed using gene DNAH5 on chromosome 5. The most significant association in DNAH5, using build 36, was obtained with a SNP corresponding to SEQ ID 5001 (see Table below for conversion to the specific DNA genotypes used). We selected a set of risk genotypes for conditional analyses. The set consisted of genotypes 1/2 and 2/2. Using this risk set, we partitioned the cases into two groups; the first group consisting of those cases that were carrier of a risk genotype and the second group consisting of the remaining cases, the non-carriers. The resulting sample sizes were respectively 461 and 50. LDSTAT (v2.0) was run in each group and a region showing association with endometriosis is reported in Table 14.1. A region associated with endometriosis in the group of carriers (has_DNAH5-1_cr) indicates the presence of an epistatic interaction between risk factors in the region and risk factors in DNAH5 (Table 14.2).
[000304] A fourth conditional analysis was performed using gene SYNE1 on chromosome 6. The most significant association signal in SYNE1 , using build 36, was obtained with a SNP corresponding to SEQ ID 5106 (see Table below for conversion to the specific DNA alleles used). We selected a set of risk genotypes for conditional analyses. The set consisted of genotypes 1/1 and 1/2. Using this risk set, we partitioned the cases into two groups; the first group consisting of those cases that were carrier of a risk genotype and the second group consisting of the remaining cases, the non-carriers. The resulting sample sizes were respectively 214 and 297. LDSTAT (v2.0) was run in each group and regions showing association with endometriosis are reported in Table 15.1 for the group of carriers and in Table 16.1 for the group of non-carriers. Regions associated with endometriosis in the group of carriers (has_SYNE1-1_cr) indicate the presence of an epistatic interaction between risk factors in the region and risk factors in SYNE1 (Table 15.2). A region associated with endometriosis in the group of non-carriers (not_SYNE1-1_cr) indicates the existence of risk factors acting independently of SYNE1 (Table 16.2).
[000305] For each region that was associated with endometriosis in the conditional analyses, we report in Tables 12.2, 13.2, 14.2, 15.2 and 16.2 the allele frequency and the relative risk (RR) for each SNP in the region. For a given SNP, the association with endometriosis was evaluated with a Chi-Square test by comparing the allele frequency in the cases with the allele frequency in the controls. Alleles with a relative risk greater than one increase the risk of developing endometriosis while alleles with a relative risk less than one are protective and decrease the risk. DNA alleles used in haplotypes (PRKCE)
SeqID 4519
Genotypes A/C
12 AC
22 CC
DNA alleles used in haplotypes (RAFl)
SeqID 4676
Genotypes T/G
12 TG
22 GG
DNA alleles used in haplotypes (DNAH5)
DNA alleles used in haplotypes (SYNEl)
6. Sub-phenotype analysis
[000306] Association analysis was performed on the full cohort and on specific subsets (6 subphenotype analyses) of the dataset corresponding to specific subtypes. The subtypes used are listed below along with the number of samples used for the analysis:
• Stage III and Stage IV combined
• Women with history of infertility
• Women with no history of infertility
• Absence of chronic pelvic pain
• Presence of ovarian cysts • Absence of ovarian cysts
[000307] Stage of disease progression was determined during the course of surgical procedures using the American Society for Reproductive Medicine (ASRM) standards. A history of infertility was defined as failure to conceive after attempting to conceive for 12 months or longer. Chronic pain and the presence of ovarian cysts were diagnosed by medical clinicians. Tables 6.1-11.1 lists the results for association analysis using LDSTATs (v2.0 and v4.0) for the regions described above based on the genome wide scan genotype data from the various subtypes. For each one of these regions, we report in Tables 6.2-11.2 the allele frequencies and the relative risk (RR) for the haplotypes contributing to the best signal at each SNP in the region.
Example 4: Gene identification and characterization
[000308] A series of gene characterization was performed for each candidate region described in Table 1. Any gene or EST mapping to the interval based on public map data or proprietary map data was considered as a candidate ENDOMETRIOSIS disease gene. The approach used to identify all genes located in the critical regions is described below.
Public gene mining
[000309] Once regions were identified using the analyses described above, a series of public data mining efforts were undertaken, with the aim of identifying all genes located within the critical intervals as well as their respective structural elements (Ae., promoters and other regulatory elements, UTRs, exons and splice sites). The initial analysis relied on annotation information stored in public databases (e.g. NCBI, UCSC Genome Bioinformatics, Entrez Human Genome Browser, OMIM - see below for database URL information). Table 2 lists the genes that have been mapped to the candidate regions. [000310] For some genes the available public annotation was extensive, whereas for others very little was known about a gene's function. Customized analysis was therefore performed to characterize genes that corresponded to this latter class. Importantly, the presence of rare splice variants and artifactual ESTs was carefully evaluated. Subsequent cluster analysis of novel ESTs provided an indication of additional gene content in some cases. The resulting clusters were graphically displayed against the genomic sequence, providing indications of separate clusters that may contribute to the same gene, thereby facilitating development of confirmatory experiments in the laboratory. While much of this information was available in the public domain, the customized analysis performed revealed additional information not immediately apparent from the public genome browsers.
[000311] A unique consensus sequence was constructed for each splice variant and a trained reviewer assessed each alignment. This assessment included examination of all putative splice junctions for consensus splice donor/acceptor sequences, putative start codons, consensus Kozak sequences and upstream in-frame stops, and the location of polyadenylation signals. In addition, conserved noncoding sequences (CNSs) that could potentially be involved in regulatory functions were included as important information for each gene. The genomic reference and exon sequences were then archived for future reference. A master assembly that included all splice variants, exons and the genomic structure was used in subsequent analyses (i.e., analysis of polymorphisms). Table 3 lists gene clusters based on the publicly available EST and cDNA clustering algorithm, ECGene.
[000312] An important component of these efforts was the ability to visualize and store the results of the data mining efforts. A customized version of the highly versatile genome browser GBrowse (http://www.gmod.org/) was implemented in order to permit the visualization of several types of information against the corresponding genomic sequence. In addition, the results of the statistical analyses were plotted against the genomic interval, thereby greatly facilitating focused analysis of gene content. Computational Analysis of Genes and GeneMaps
[000313] In order to assist in the prioritization of candidate genes for which minimal annotation existed, a series of computational analyses were performed that included basic BLAST searches and alignments to identify related genes. In some cases this provided an indication of potential function. In addition, protein domains and motifs were identified that further assisted in the understanding of potential function, as well as predicted cellular localization.
[000314] A comprehensive review of the public literature was also performed in order to facilitate identification of information regarding the potential role of candidate genes in the pathophysiology of ENDOMETRIOSIS disease. In addition to the standard review of the literature, public resources (Medline and other online databases) were also mined for information regarding the involvement of candidate genes in specific signaling pathways. A variety of pathway and yeast two hybrid databases were mined for information regarding protein-protein interactions. These included BIND, MINT, DIP, Interdom, and Reactome, among others. By identifying homologues of genes in the ENDOMETRIOSIS disease candidate regions and exploring whether interacting proteins had been identified already, knowledge regarding the GeneMaps for ENDOMETRIOSIS disease was advanced. The pathway information gained from the use of these resources was also integrated with the literature review efforts, as described above.
[000315] Genes identified in the WGAS and subsequent studies for ENDOMETRIOSIS disease (ENDOMETRIOSIS) were evaluated using the Ingenuity Pathway Analysis application (IPA, Ingenuity systems) in order to identify direct biological interactions between these genes, and also to identify molecular regulators acting on those genes (indirect interactions) that could be also involved in ENDOMETRIOSIS. The purpose of this effort was to decipher the molecules involved in contributing to ENDOMETRIOSIS. These gene interaction networks are very valuable tools in the sense that they facilitate extension of the map of gene products that could represent potential drug targets for ENDOMETRIOSIS.
ENDOMETRIOSIS Genemap and Pathways
[000316] The GWAS and subsequent data mining analyses resulted in a GeneMap that contains networks highly relevant to ENDOMETRIOSIS as well as many genes under hormonal control. The following examples of the emerging GeneMaps includes signaling pathways in cell proliferation, apoptosis, cell cycle, cell communication, cell structure, motility and hormonal regulation. Many of the identified regions contain genes involved in biologically relevant pathways, or associated conditions such as an oncogenesis-like mechanism, angiogenesis and infertility.
[000317] Signaling pathway; It has already been suggested based on expression studies that the RAS/RAF/MAPK and PI3K pathways may be involved in initial ENDOMETRIOSIS development and pathophysiology. Among genes discovered in the GWAS that relates and confirms these observations are, RAF1 , PRKCE, PRKD1 , RALGPS1 and PIK3C2A (Stage III/IV subphenotype). These genes play roles in a multitude of pathways including cell proliferation, cell differentiation, survival/apoptosis, cell cycle, development, cytoskeleton, angiogenesis, transformation and invasion/locomotion. Moreover, the PRKCE gene has been shown to be over-expressed in ectopic endometrium as compared to eutopic tissue. All of these are biologically relevant for ENDOMETRIOSIS.
[000318] Cell proliferation / Angiogenesis: Angiogenesis might also play an important role in the pathogenesis of ENDOMETRIOSIS. It is viewed as a major prerequisite for the initiation and progression of the disease: known role in the survival of the implants and the development of ENDOMETRIOSIS. Anti- angiogenic agents may provide a novel therapeutic approach for the treatment of ENDOMETRIOSIS. The genes from the observed GWAS results herein that may explain the Angiogenesis connection are RAF1 , PPARG, PRKCE, PRKD1 , PIK3C2A (Stage III/IV subphenotype) and SMOC2 (Not lnfertlity subphenotype).
[000319] Hormonal regulation; ENDOMETRIOSIS is an estrogen-dependent disease and it is known that treatments tend to suppress estrogen synthesis. Several of the identified pathways include genes that are regulated by or involved in the regulation of estrogen signaling: RAF1 , PRKCE, KCNQ3, AVPR2 (from conditional, epistatic to SYNE1) and ACE2 (from conditional, heterogeneity to SYNE1).
[000320] Cell Structure and Motility (Cytoskeleton): Active cell proliferation necessitates constant reorganization of the cytoskeleton. In ENDOMETRIOSIS there is adherence of endometrial cells to ectopic locations. Cell motility in also involved in ENDOMETRIOSIS because of migration and transport of endometrial tissue to ectopic locations. We have identified several cell adhesion molecules that could be involved in cell-cell or cell-matrix interaction necessitated in adherence to ectopic locations. RALGPS1 , PRKD1 and PACSIN2 (from conditional, epistatic to SYNE1) are GWAS genes that are known to have a role in cell proliferation and cytoskeleton remodeling. SYNE1 , KCNQ3, PRICKLE1 and SLC8A1 (from conditional, epistatic to SYNE1) are GWAS genes involved in cell structure. PPFIBP1 (Stage III/IV subphenotype), is involved in focal adhesions, tumor invasiveness and metastasis.
[000321] Infertility: Kinetics between endometrial/fallopian ciliated cells and uterine contractions may be important for normal function of fertilization and menstruation cycle. Two of the genes, DNAH5 and DNAHL1 code for cilia motor proteins. A study on ultrastructural aspects of endometrium in infertile women with septate uterus have shown irregular nonciliated cells with rare microvilli, incomplete ciliogenesis on ciliated cells, and decrease in the ciliated:nonciliated cell ratio.
[000322] Oncoqenesis-like mechanism: Although ENDOMETRIOSIS is not a cancer, molecular and/or regulatory mechanisms responsible for the development of the disease may be similar. From the analyses of the GWAS data, it was found that the downstream genes that may explain the oncogenesis connection of ENDOMETRIOSIS include RAF1 , MCM3AP, MAD2L2 and H2AFY. RAF 1 is an oncogene while the other genes are respectively implicated in DNA replication, cell division and gene silencing, all activities that are known to be important in the development of tumorogenesis.
ENDOMETRIOSIS and drug targets
[000323] Considering the fact that angiogenesis might play an important role in the pathogenesis of ENDOMETRIOSIS, anti-angiogenic factors are used as an experimental treatment in animal models. They have been shown to cause regression and/or inhibition of the growth of endometriotic lesions. Most antiangiogenic agents have been discovered by identifying endogenous molecules that inhibit endothelial cells growth. This traditional approach has produced a number of anti-angiogenics; platelet factor-4 (PF4), thrombospondin, tumour necrosis factor (TNF)-a, interferon-c-inducible protein-10 (IP-10), angiostatin, endostatin, vasostatin, bactericidal-permeability increasing protein (BPI). About 40 anti-angiogenic agents, identified using various approaches, are currently known. These drugs are relatively safe; some compounds affect normal reproductive functions but most are safe. VEGF has a known role in angiogenesis. Formation of endometriotic lesions is significantly impaired with anti-hVEGF antibody. Anti-angiogenic agents may provide a novel therapeutic approach for the treatment of ENDOMETRIOSIS.
[000324] PPARG is one of the genes identified on our GWAS study. PPARG is a member of the peroxisome proliferator-activated receptor subfamily of nuclear receptors. PPARG is a regulator of adipocyte differentiation and has been implicated in the pathology of numerous diseases including obesity, diabetes, atherosclerosis and cancer. Thiazolidinediones (TZD) are artificial ligands of PPARs, and are used clinically as anti-diabetic drugs. It has been shown that PPAR-α and -v are expressed by peritoneal macrophages isolated from ENDOMETRIOSIS patients. Also, the PPARG ligand rosiglitazone inhibit angiogenesis in tumors. This is relevant for ENDOMETRIOSIS. [000325] ENDOMETRIOSIS is an estrogen-dependent disease. Endometriotic implants are dependent on estrogen for their maintenance and growth. Treatments tend to suppress estrogen synthesis. In the examples of Genemaps described above, several of the identified pathways include genes that are regulated by or involved in the regulation of estrogen signaling. Examples of such genes are PRKCE, RAF1 , KCNQ3 and AVPR2 (from conditional analysis herein). Also numerous network genes in the described Genemaps are targeted by estrogen. These are IQGAP1 , SRC, EP300, SP1 , E2F1 , MAP2K1 , HEXIM1 , NTS, OXT and OXTR.
[000326] In the signaling pathway of the described Genemap, many compounds target GWAS identified genes or network genes. Recent breakthroughs in molecular oncology have identified a number of critical downstream signaling cascades that are activated by tyrosine kinases implicated in the development of cancer. Compounds are focused on inhibiting these critical downstream pathways, which include the PI3K, RAS/RAF/MAPK cascades (all genes identified in the GWA study, are also found in the described Genemap). For example, sorafenib (Nexavar) is a known drug that target one our top hits, RAF1. Sorafenib is an anticancer medicine used to treat adults with kidney cancer called advanced renal carcinoma. Another compound that specifically targets RAF1 is XL281. Phase 1 trial in patients with advanced solid tumors is ongoing. Similar compound is XL147, which selectively targets PI3K. A Phase 1 trial in patients with solid tumors is ongoing. Also drugs that are PKC inhibitor, which inhibit PRKCE and / or PRKD1 (both genes identified in the GWA study), are used for various indications. MAPK inhibitors (MAP2K1 is a network gene) are also drugs tested for multiple tumors and advanced cancers.
[000327] Another network gene in the described Genemap is EP300, a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation. EP300 is a target for histone deacetylase inhibitors, and is used in cancer therapy. [000328] Thus, in the described genome wide association study (GWAS), many of the genes are druggable and biologically relevant for ENDOMETRIOSIS.
Expression Studies
[000329] In order to determine the expression patterns for genes, relevant information was first extracted from public databases. The UniGene database, for example, contains information regarding the tissue source for ESTs and cDNAs contributing to individual clusters. This information was extracted and summarized to provide an indication in which tissues the gene was expressed. Particular emphasis was placed on annotating the tissue source for bona fide ESTs, since many ESTs mapped to Unigene clusters are artifactual. In addition, SAGE and microarray data, also curated at NCBI (Gene Expression Omnibus), provided information on expression profiles for individual genes. Particular emphasis was placed on identifying genes that were expressed in tissues known to be involved in the pathophysiology of endometriosis. To complement available information about the expression pattern of candidate disease genes, two experimental approaches were used. The first one was a RT-PCR based semiquantitative gene expression profiling method that could be applied to a large number of target sequences (genes, transcripts, ESTs) over a panel of 24 selected tissues. The second was to map expression sites of mouse transcripts orthologous to a small set of human disease candidate genes in the mouse embryo (day 10.5, 12.5 and 15.5), in the postnatal stages (day 1 and 10) and at adulthood using in situ hybridization (ISH) method.
Semi-quantitative gene expression profiling by RT-PCR
[000330] Total human RNA samples from 24 different tissues Total RNA sample were purchased from commercial sources (Clontech, Stratagene) and used as templates for first-strand cDNA synthesis with the High-Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions. A standard PCR protocol was used to amplify genes of interest from the original sample (50 ng cDNA); three serial dilutions of the cDNA samples corresponding to 5, 0.5 and 0.05 ng of cDNA were also tested. PCR products were separated by electrophoresis on a 96-well agarose gel containing ethidium bromide followed by UV imaging. The serial dilutions of the cDNA provided semi-quantitative determination of relative mRNA abundance. Tissue expression profiles were analyzed using standard gel imaging software (Alphalmager 2200); mRNA abundance was interpreted according to the presence of a PCR product in one or more of the cDNA sample dilutions used for amplification. For example, a PCR product present in all the cDNA dilutions (i.e. from 50 to 0.05 ng cDNA) was designated ++++ while a PCR product only detectable in the original undiluted cDNA sample (i.e., 50 ng cDNA) was designated as + or +/-, for barely detectable PCR products (see Table 17). For each target gene, one or more gene-specific primer pairs were designed to span at least one intron when possible. Multiple primer-pairs targeting the same gene allowed comparison of the tissue expression profiles and controlled for cases of poor amplification.
in situ hybridization (ISH) study General procedure:
[000331] 4 genes, highlighted in the GWAS study (full sample and has ovarian cyct subphenotype dataset), namely H2afy, Mad2l2, Mcm3ap and Nrxni , were selected for further characterization by ISH in mouse. For each gene, a fragment of the mouse ortholog cDNA was use for the synthesis of cRNA probes (Table 18). To maximally preserve the integrity of tissue in its environment, mouse whole-body sections were used (Figure D). Whole bodies were frozen cut into 10-μm sections. To complement the whole-body sections, tissue arrays including reproductive organs (RO), general tissue array (TA) and brain array (BA) were used (Figure D). Tissue slices were mounted on glass microscope slides, fixed in formaldehyde and hybridized with 35S-labeled cRNA probes. Antisense cRNA generated positive signals whereas sense cRNA (identical to mRNAs) generated negative (control) signals. Prior to gene-specific ISH, the tissues were validated with riboprobes to LDL receptor mRNA (data not shown). Following ISH, gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with appropriate exposure times.
Detailed procedure:
Mouse cDNA clone and DNA templates preparation
[000332] cDNA clones of mouse orthologs to human genes H2afy, Mad2l2, Mcm3AP and Nrxni were obtained from commercial source (Open Biosystem). DNA fragments to be used as templates for the cRNA probes synthesis were amplified by PCR and cloned into pGEM-7Zf(+)/LIC-F (ATCC #87048). After sequence validation, the templates for the antisense cRNA probes synthesis were generated by PCR using forward primers located at the 5' end of the cloned DNA fragments and a reverse primer located upstream of the SP6 polymerase promoter (in the vector). Similarly, the templates for the sense (control) cRNA probes synthesis were generated by PCR using a forward primer located upstream of the T7 promoter (in the vector) and reverse primers located at the 3' end of the cloned DNA fragments.
cRNA probe preparation
[000333] cRNA transcripts were synthesized in vitro from linear DNA fragments by run-off transcription with the SP6 or T7 RNA Polymerase from their respective promoters. Cold probe synthesis proved that DNA templates are functional and, hence, applied to radioactive probe synthesis labeled with 35S- UTP (>1 ,000 Ci/mmol; Amersham).
Tissues preparation.
[000334] Tissues were frozen-cut into 10-μm sections, mounted on gelatin- coated slides and stored at -80°C. Before ISH, they were fixed in 4% formaldehyde (freshly made from paraformaldehyde) in phosphate-buffered saline (PBS), treated with triethanolamine/acetic anhydride, washed and dehydrated with a series of ethanol. Hybridization and washing procedures.
[000335] Sections were hybridized overnight at 55°C in 50% deionized formamide, 0.3 M NaCI, 20 mM Tris-HCI, pH 7.4, 5 mM EDTA, 10 nM NaPO4, 10% dextran sulfate, 1 x Denhardt's, 50 μg/ml total yeast RNA, and 50-80,000 cpm/μl 35S-labeled cRNA probe. The tissue was subjected to stringent washing at 65°C in 50% formamide, 2 x SSC, and 10 mM DTT, followed by washing in PBS before treatment with 20 μg/ml RNAse A at 37°C for 30 minutes. After washes in 2 x SSC and 0.1 x SSC for 10 minutes at 37°C, the slides were dehydrated, apposed to X-ray film for 5 days, then dipped in Kodak NTB nuclear track emulsion, and exposed for 12 days in light-tight boxes with desiccant at 4°C.
Imaging.
[000336] Photographic development was undertaken with Kodak D-19. The slides were lightly counterstained with cresyl violet and analyzed under both light- and darkfield optics. Sense control cRNA probes (identical to mRNAs) always gave background levels of the hybridization signal.
Storage and rehydration
[000337] "Crystallization" of any section could be repaired by allowing the coverslips to fall off after soaking in xylene for 24-48 hours. The slides were rehydrated to 70% EtOH and then re-dehydrated again in a series of ethanol (80%, 96% and 2 x 100% for 2 minutes each). After 3 changes with xylene, the coverslips were mounted with Cytoseal (VWR Scientific) or other comparable mounting medium. Using the same method, the coverslips were removed for histological staining to take brightfield micrographs. Histological stains that require acidic conditions could dissolve silver grains. Overstaining could obscure the silver grains. Any excess mounting medium or residual emulsion on the back of the slides was removed with a single-edged razor. The re-coverslipped slides were dried flat for 24 hours, and stored indefinitely at room temperature. Viewing original slides
[000338] The results are best viewed by darkfield illumination, with x2.5, x4, x10, x25 and 4Ox objectives; the silver grains can be localized over particular cells. The antisense probe detects mRNA, and the sense control probe shows the background level of silver grains for the experiments.
Results:
H2afy
[000339] Following ISH, H2afy gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 3 days and 12 days, respectively. Results are presented in Tables 19 and 20 and Figures E to N.
[000340] Analysis of ISH results provide evidence for H2afy mRNA presence in all embryonic stages studied (Figure E). H2afy displays a widespread if not ubiquitous distribution pattern in the midgestation stages e10.5, e12.5 and e15.5. At birth, H2afy mRNA distribution pattern shows differentiation in high and low expression sites, to form a mosaic like pattern later at adulthood. More or less pronounced hybridization labeling occurs in the central nervous system, pituitary gland, adrenal gland, thymus, spleen, lymph nodes, testis, ovary and uterus. The later, in pregnant female displays hybridization in the endometrium and decidua containing embryonic origin trophoblasts. Complete picture of H2afy mRNA distribution in the adult mouse is shown in Table 19.
[000341] In conclusion, H2afy belongs to a class of ubiquitously expressed genes in the embryonic mouse which over a postnatal developmental differentiation process, acquire a cell and tissue specific pattern of distribution. This expression profil suggest that H2afy may play a role in both developmental and adulthood functions, including nervous, endocrine, immune and reproductive functions. Table 19: Detection of H2AFY mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
Average labeling level - = not detectable, + = very weak, ++ = weak, +++ = medium, and ++++ = high and +++++ = very high H2AFY mRNA concentration Abbreviation CNS = central nervous system, PNS = peripheral nervous system
Table 20: H2AFY mRNA tissue distribution in the adult mouse
Cl = solely
Mad2l2
[000342] Following ISH, Mad2l2 gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 4 days and 14 days, respectively. Results are presented in Tables 21 and 22 and Figures O to S.
[000343] Analysis of ISH results provide evidence for Mad2l2 mRNA presence in all embryonic stages studied, including e10.5, e12.5, and e15.5 (Figure O). Mad2l2 seems to be mostly expressed in the primordium of the nervous system, where neuronal expression patterns remain elevated until the end of the intrauterine life, followed by dramatic decline on postnatal day 10 and adulthood. Exception is the cerebellum, which continues its development after birth, displaying postnatal pattern of hybridization. Still, Mad2l2 mRNA concentration declines at adult stage. Whenever embryonic in majority, or postnatal in the cerebellar region particularly, Mad2l2 activity seems likely to relate to the formation of the embryonic nervous system. Once formed, the adult brain and spinal cord synthesize much less Mad2l2 mRNA. Similar process has been observed in the peripheral nervous system sensory ganglia (dorsal root ganglia), ortosympathetic ganglia (paravertebral ganglia), olfactory neuroepithelium, retina in the eye and the organ of Corti in the ear, all displaying strong hybridization signal in p1 mice but weak in the adulthood. [000344] In contrast to the developmental mice, in the adulthood, low Mad2l2 expression levels were evident in most tissues, as shown in Table 21. Highest Mad2l2 mRNA levels were detected in the male testis seminiferous tubules. In the female, the ovary and uterus contained low concentrations Mad2l2 mRNA. In contrast, pregnant mouse uterine tissue examined on day 5.5 and 7.5 post- coitum, displayed increased levels of Mad2l2 mRNA around the sites of the conceptus implantation, where a decidua is formed. This observation suggests an induction of Mad2l2 expression specifically involved in the implantation or post- implantation processes. Finally, the liver, spleen and kidney displayed low Mad2l2 levels.
[000345] Comparison of Mad2l2 ontogeny expression patterns with that of PCNA provides evidence of their partial overlapping. Thus, in p1 mice high concentrations of Mad2l2 mRNA occur in the olfactory neuroepithelium, cerebellum, dorsal root ganglia, paravertebral ganglia, kidney marginal zone and intestine this correlation is evident (Table 22). In the adult mice, high level overlapping occurs in the testis and a pregnant mice uterus. Mad2l2 / PCNA overlapping expression patterns suggests a link with cell proliferative / periproliferative events (proliferation arrest, apoptosis, cell migration). Still, in some regions of the embryonic and newborn mice, this correlation appears of a low degree, suggesting selectivity. For example, low correlation appears to occur between the brain (high Mad2l2 and low PCNA mRNA levels) or liver (low Mad2l2 and high PCNA mRNA levels). It is, thus, possible that Mad2l2 brings the specificity to the tissue and cell peri-proliferative processes.
[000346] In conclusion high level Mad2l2 expression was documented in the developing central and peripheral nervous system, but down-regulated to the adulthood. In the late stage, following a postnatal up-regulation, high mad2l2 mRNA concentration occurs in the testis, whereas following gene induction, increased Mad2l2 expression levels are evident in the pregnant mouse uterus, both processes suggesting Mad2l2 role in the reproductive events. Partially overlapping ontogeny patterns with that of PCNA suggest mad2l2 functional involvement in the tissue- and cell-specific peri-proliferative events in the mouse development and reproduction.
Table 21 : Detection of MAD2L2 mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
Average labeling level: - = not detectable; + = very weak; ++ = weak; +++ = medium; and ++++ = high and +++++ = very high MAD2L2 mRNA concentration. Abbreviations: CNS = central nervous system; PNS = peripheral nervous system.
Table 22: Relative Correlation Between MAD2L2 and PCNA mRNA Ontogeny Distribution Patterns. Scale as in Table 21.
Mcm3ap
[000347] Following ISH, Mcm3ap gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 5 days and 17 days, respectively. Results are presented in Tables 23 and 24 and Figures T to W.
[000348] Analysis of ISH results provide evidence for a Mcm3ap expression at low-level in the embryonic stages studied (Figure T). ISH results were readable following 5-day exposure of X-Ray Films, which is a limit of mRNA detection by a technique. Mcm3ap displays a widespread if not ubiquitous distribution pattern observed from the midgestation stages e10.5, e12.5 and e15.5 to the adulthood with no significant changes in the pattern of tissue specificity and mRNA concentration. Although slightly elevated hybridization levels were observed in some tissues such as the thymus and brain regions such as cerebellum and hippocampus of the newborn, postnatal and adult mice, these tissues are characterized by locally high density of cells. Thus, mcm3ap concentration in these structures reflected rather the increasing cell density than gene expression regulation mechanism. The overview of mcm3ap mRNA distribution pattern in the adult mouse is shown in Table 24.
[000349] In conclusion, Mcm3ap belongs to a class of low-level ubiquitously expressed genes that maintain their mRNA distribution pattern and concentration level spanning over a life, in the mouse from the embryonic stages to the adulthood. Mcm3ap represents likely a housekeeping class of the genes.
Table 23: Detection of MCM3AP mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
Average labeling level: - = not detectable; + = very weak; ++ = weak; +++ = medium; and ++++ = high and +++++ = very high GENE7 mRNA concentration.
Table 24: MCM3AP mRNA tissue distribution in the adult mouse
Scale: - = not detectable; + = weak; ++ = intermediate; +++ = medium; ++++ = strong and +++++ = very strong labelling; Cl = criteria insufficient to identify cell type at present condition.*; NE = not examined. *As the cell types were solely established based on their topography and morphology they are considered as presumptive only. Specific phenotype markers are required to identify cell type unambiguously.
Nrxni
[000350] Following ISH, Nrxni gene expression patterns were analyzed by both x-ray film autoradiography and emulsion autoradiography with exposure times of 2 days and 10 days, respectively. Results are presented in Tables 25 and 26 and Figures X to CC.
[000351] Analysis of ISH results provide evidence for a Nrxni expression generally at high-level in the embryonic, newborn postnatal and adult mouse stages Table 25 and Figure X. Not detectable on day 10.5, ISH signal was evident on day 12.5 in the rudimental central (CNS) and peripheral (PNS) nervous system, persisting elevated along further developmental stages. The highest level of expression was noted to occur in CNS and PNS on postnatal day 10, followed by decline in the adult stage. Nrxni distribution in the adult stage is summarized in the Table 26. Briefly, presence of Nrxn1 mRNA was confined to neuronal cells of the grey matter. There was not detectable Nrxni mRNA in the white matter cells with oligodendrocyte topography. Important to note is that the majority, but not all neuronal cells displayed Nrxni mRNA labeling. For example, there was not labeling in the Purkinje cells of the cerebellar folia and few other discreet regions but not shown here. For the above reason, Nrxni distribution cannot be defined as pan-neuronal, but rather neuron-specific.
[000352] To scrutinize Nrxni mRNA-labeled neurons in PNS ganglia the newborn and postnatal stages (p1 and p10) appeared especially useful when compared to adult stage: (1) there were higher gene expression levels evident in pp1 and p10 ganglia and (2) higher choice of sections that passed throughout relevant regions in comparison to low choice in the adult stage sections selection. A list of Nrxni-labeled PNS ganglia include the sensory cranial ganglia such as the trigeminal ganglion as well as dorsal root ganglia. The ganglia of the sympathetic nervous system and visceral microganglia contributing to the plexus Auerbach expressed Nrxni mRNA. In addition to CNS and PNS, the endocrine glands such as the pituitary gland and adrenal medulla displayed a low to medium Nrxni mRNA concentrations.
[000353] In conclusion, Nrxni belongs to a class of high-level neuronal- specific genes with distribution pattern following most CNS and PNS regions and two endocrine glands. In CNS and PNS, Nrxni occurs at concentrations that are up-regulated postnatally to a maximal levels measured on day 10. Nrxni represents likely a good neuronal marker, especially to the plexus Auerbach in the gut.
Table 25. Detection of NRXN1 mRNA in whole body sections from 3 different mouse ontogeny stages, 2 postnatal stages and adulthood
Average labeling level: - = not detectable; + = very weak; ++ = weak; +++ = medium; and ++++ = high and +++++ = very high GENE9 mRNA concentration.
Table 26. NRXN1 mRNA tissue distribution in the adult mouse
Scale: - = not detectable; + = weak; ++ = intermediate; +++ = medium; ++++ = strong and +++++ = very strong labelling; Cl = criteria insufficient to identify cell type at present condition.*; NE = not examined.
*As the cell types were solely established based on their topography and morphology they are considered as presumptive only. Specific phenotype markers are required to identify cell type unambiguously.

Claims

WE CLAIM:
1. A method of constructing a GeneMap for ENDOMETRIOSIS disease comprising identifying at least two chromosomal loci associated with ENDOMETRIOSIS disease, wherein said at least two chromosomal loci are selected from the genomic regions listed in Table 1.
2. The method of claim 1 , wherein said population is a general population.
3. The method of claim 1 , wherein said population is a founder population.
4. The method of claim 3, wherein said founder population is the population of Quebec.
5. The method of claim 1 , wherein said at least two chromosomal regions are selected from the genes in Table 2, 3 or 4.
6. The method of claim 5, wherein said genes are used to construct gene networks based on the functional relationship of gene products interactions.
7. The method of claim 6, wherein the interactions are direct, indirect, or a combination thereof.
8. The method of claim 1 , wherein the identifying comprises screening for the presence or absence of at least one single nucleotide polymorphisms (SNPs) from Tables 5-16 in at least one sample.
9. The method of claim 8, wherein the screening comprises the steps of: (a) obtaining biological samples from at least one disease patient; (b) screening for the presence or absence of at least one SNP or a group of SNPs from Tables 5-16 within each biological sample; and (c) evaluating whether said SNP or a group of SNPs shows a statistically significant skewed genotype distribution between a group of patients compared to a control.
10. The method of claim 9, wherein said biological samples are fluid, serum, tissue or buccal swabs, saliva, mucus, urine, stools, vaginal secretions, lymph, amiotic liquid, pleural liquid or tears.
11. The method of claim 9, wherein said patients and controls are from a human population.
12. The method of claim 11 , wherein said patients and controls are recruited independently according to specific phenotypic criteria.
13. The method of claim 11 , wherein said patients and controls are recruited in the form of trios comprising two parents and one child.
14. The method of claim 8, wherein said screening is performed by a method selected from the group consisting of an allele-specific hybridization assay, an oligonucleotide ligation assay, an allele-specific elongation/ligation assay, an allele-specific amplification assay, a single-base extension assay, a molecular inversion probe assay, an invasive cleavage assay, a selective termination assay, RFLP, a sequencing assay, SSCP, a mismatch-cleaving assay, and denaturing gradient gel electrophoresis.
15. The method of claim 8, wherein said screening is carried out on each individual of a cohort at each of at least one SNP or a group of SNPs from Tables 5-16.
16. The method of claim 8, wherein said screening is carried out on pools of patients and pools of controls.
17. The method of claim 8, wherein the genotype distribution is compared one SNP at a time.
18. The method of claim 8, wherein the genotype distribution is compared with a group of markers from Tables 5-16 forming a haplotype.
19. The method of claim 17, wherein the genotype distribution is compared using the allelic frequencies between the patient pools and control pools.
20. The method of claim 1 , wherein the GeneMap comprises all of the genes of Tables 2-4.
21. A method of diagnosing ENDOMETRIOSIS disease, the predisposition to ENDOMETRIOSIS disease, or the progression or prognostication of ENDOMETRIOSIS disease, comprising determining the amount and/or concentration of at least one polypeptide from Tables 2-4 and/or at least one nucleic acid encoding the polypeptide present in said biological sample
22. The method of claim 21 , wherein the diagnosing comprises the steps of: (a) obtaining a biological sample of mammalian body fluid or tissue to be diagnosed; (b) comparing the amount and/or concentration of said polypeptide and/or nucleic acid encoding the polypeptide determine in said biological sample with the amount and/or concentration of said polypeptide and/or nucleic acid encoding the polypeptide as determined in a control sample, wherein the difference in the amount of said polypeptide and/or nucleic acid encoding the polypeptide is indicative of ENDOMETRIOSIS disease or the stage of ENDOMETRIOSIS disease.
23. The method of claim 21 , wherein a nucleic acid probe is used for determining the amount and/or concentration of at least one nucleic acid sequence from Tables 2-4 encoding the polypeptide.
24. The method of claim 23, wherein said nucleic acid probe is selected from the nucleic acid sequences designated as SEQ ID NO: 1 to 6723.
25. The method of claim 23, wherein said nucleic acid probe comprises nucleic acids hybridizing to the nucleic acid sequences designated as SEQ ID NO: 1 to 6723, and/or fragments thereof.
26. The method of claim 23, wherein said nucleic acid probe comprises nucleic acids hybridizing to at least five nucleic acid sequences from Table 2, 3 or 4.
27. The method of claim 23, wherein said nucleic acid probe specifically hybridizes to at least 10 nucleic acid sequences from Tables 2-4.
28. The method of claim 23, wherein said nucleic acid probe specifically hybridizes to at least 20 nucleic acid sequences from Tables 2-4.
29. The method of claim 23, wherein said nucleic acid probe specifically hybridizes to at least 50 nucleic acid sequences from Tables 2-4.
30. The method of claim 23, wherein said nucleic acid probe specifically hybridizes to at least 100 nucleic acid sequences from Tables 2-4.
31. The method of claim 23, wherein said nucleic acid probe specifically hybridizes to at least 100 nucleic acid sequences from Tables 2-4.
32. The method of claim 23, wherein said nucleic acid probe is at least about 10 nucleotides in length.
33. The method of claim 23, wherein said nucleic acid probe is at least about 30 nucleotides in length.
34. The method of claim 23, wherein said nucleic acid probe is at least about 50 nucleotides in length.
35. The method of claim 23, wherein a PCR technique is used for determining the amount and/or concentration of at least one nucleic acid from Tables 2-4.
36. The method of claim 21 , wherein a specific antibody is used for determining the amount and/or concentration of at least one polypeptide from Tables 2-4.
37. The method of claim 36 wherein said antibody is selected from the group comprising polyclonal antiserum, polyclonal antibody, monoclonal antibody, antibody fragments, single chain antibodies and diabodies.
38. The method of claim 21 , wherein the amounts and/or concentrations of at least five polypeptides or nucleic acids are determined.
39. A method of detecting susceptibility to ENDOMETRIOSIS disease comprising detecting at least one mutation or polymorphism in the nucleic acid molecule selected from Tables 2-4 in a patient.
40. The method of claim 39, wherein said method comprises hybridizing a probe to said patient's sample of DNA or RNA under stringent conditions which allow hybridization of said probe to nucleic acid comprising said mutation or polymorphism, wherein the presence of a hybridization signal indicates the presence of said mutation or polymorphism in at least one gene from Tables 2-4.
41. The method of claim 39, wherein the patient's DNA or RNA has been amplified and said amplified DNA or RNA is hybridized.
42. The method of claim 39, wherein said method comprises using a single-stranded conformation polymorphism technique to assay for said mutation.
43. The method of claim 39, wherein said method comprises sequencing at least one gene from Tables 2-4 in a sample of RNA or DNA from a patient.
44. The method of claim 39, wherein said method comprises determining the sequence of at least one gene from Tables 2-4 by preparing cDNA from RNA taken from said patient and sequencing said cDNA to determine the presence or absence of a mutation.
45. The method of claim 39, wherein said method comprises performing an RNAse assay.
46. The method of claim 39, wherein said probe is attached to a microarray or a bead.
47. The method of claim 39, wherein said probes are oligonucleotides.
48. The method of claim 40, wherein said sample is selected from the group consisting of blood, normal tissue and tumor tissue.
49. The method of claim 39, wherein the mutation is selected from the group consisting of at least one of the SNPs from Tables 5-16, alone or in combination.
50. The method of claim 21 , further comprising comparing the level of expression or activity of a polypeptide of Tables 2-4 in a test sample from a patient with the level of expression or activity of the same polypeptide in a control sample wherein a difference in the level of expression or activity between the test sample and control sample is indicative of ENDOMETRIOSIS disease.
51. A method of treatment of ENDOMETRIOSIS disease in a mammal in need thereof, comprising the steps of: performing steps a) to c) according to claim 22; and treating the mammal in need of said treatment; wherein said medical treatment is based on the stage of the disease.
52. A method of diagnosing susceptibility to ENDOMETRIOSIS disease in an individual, comprising screening for an at-risk haplotype of at least one gene or gene region from Tables 2-4, that is more frequently present in an individual susceptible to ENDOMETRIOSIS disease compared to a control individual, wherein the presence of the at-risk haplotype is indicative of a susceptibility to ENDOMETRIOSIS disease.
53. The method of claim 52 wherein the at-risk haplotype is indicative of increased risk for ENDOMETRIOSIS disease.
54. The method of claim 53, wherein the risk is increased at least about 20%.
55. The method of claim 52, wherein the at-risk haplotype is characterized by the presence of at least one single nucleotide polymorphism from Tables 5-16.
56. The method of claim 52, wherein screening for the presence of an at- risk haplotype in at least one gene from Tables 2-4, comprises enzymatic amplification of nucleic acid from said individual or amplification using universal oligos on elongation/ligation products.
57. The method of claim 56, wherein the nucleic acid is DNA.
58. The method of claim 57, wherein the DNA is human DNA.
59. The method of claim 52, wherein screening for the presence of an at- risk haplotype in at least one gene from Tables 2-4 comprises: (a) obtaining material containing nucleic acid from the individual; (b) amplifying said nucleic acid; and (c) determining the presence or absence of an at-risk haplotype in said amplified nucleic acid.
60. The method of claim 59, wherein determining the presence of an at-risk haplotype is performed by electrophoretic analysis.
61. The method of claim 59, wherein determining the presence of an at-risk haplotype is performed by restriction length polymorphism analysis.
62. The method of claim 59, wherein determining the presence of an at-risk haplotype is performed by sequence analysis.
63. The method of claim 59, wherein determining the presence of an at-risk haplotype is performed by hybridization analysis.
64. A method of diagnosing a susceptibility to ENDOMETRIOSIS disease, comprising detecting an alteration in the expression or composition of a polypeptide encoded by at least one gene from Tables 2-4 in a test sample, in comparison with the expression or composition of a polypeptide encoded by said gene in a control sample, wherein the presence of an alteration in expression or composition of the polypeptide in the test sample is indicative of a susceptibility to ENDOMETRIOSIS Disease.
65. The method of claim 64, wherein the alteration in the expression or composition of a polypeptide encoded by said gene comprises expression of a splicing variant polypeptide in a test sample that differs from a splicing variant polypeptide expressed in a control sample.
66. A drug screening assay comprising: a)administering a test compound to an animal having ENDOMETRIOSIS disease, or a cell population isolated therefrom; and (b) comparing the level of gene expression of at least one gene from Tables 2-4 in the presence of the test compound with the level of said gene expression in normal cells; wherein test compounds which provide the level of expression of one or more genes from Tables 2-4 similar to that of the normal cells are candidates for drugs to treat ENDOMETRIOSIS disease.
67. A pharmaceutical preparation for treating an animal having ENDOMETRIOSIS disease comprising a compound identified by the assay of claim 66 and a pharmaceutically acceptable excipient.
68. A method for treating an animal having ENDOMETRIOSIS disease comprising administering a compound identified by the assay of claim 66.
69. A method for predicting the efficacy of a drug for treating ENDOMETRIOSIS disease in a human patient, comprising: (a) obtaining a sample of cells from the patient; (b) obtaining a gene expression profile from the sample in the absence and presence of the drug ; the gene expression profile comprising one or more genes from Tables 2-4; and (c) comparing the gene expression profile of the sample with a reference gene expression profile, wherein similarity between the sample expression profile and the reference expression profile predicts the efficacy of the drug for treating ENDOMETRIOSIS disease in the patient.
70. The method of claim 69, further comprising exposing the sample to the drug for treating ENDOMETRIOSIS disease prior to obtaining the gene expression profile of the sample.
71. The method of claim 69, wherein the sample of cells is derived from a tissue selected from the group consisting of: the brain, reproductive system, digestive system, skin, scalp, muscle and nervous tissue.
72. The method of claim 71 , wherein the cells are selected from the group consisting of: ovarian cell, uterine cell, vaginal cell, hair cell, brain cell, muscle cell, neutrophil, dentric cell, T cell, mast cell, CD4+ lymphocyte, monocyte, macrophage, dendritic cell, and epithelial cell.
73. The method of claim 69, wherein the sample is obtained via brain biopsy.
74. The method of claim 69, wherein the gene expression profile comprises expression values for all of the genes listed in Tables 2-4.
75. The method of claim 74, wherein the gene expression profile of the sample is obtained by detecting the protein products of said genes.
76. The method of claim 69, wherein the gene expression profile of the sample is obtained using a hybridization assay to oligonucleotides contained in a microarray.
77. The method of claim 76, wherein the oligonucleotides comprises nucleic acid molecules at least 95% identical to the gene sequences from Tables 2-4.
78. The method of claim 69, wherein the reference expression profile is that of cells derived from patients that do not have ENDOMETRIOSIS disease.
79. The method of claim 69, wherein the drug is selected from the group consisting of symptom relievers.
80. The method of claim 69, wherein said patient's sample of DNA has been amplified or cloned.
81. A method for predicting the efficacy of a drug for treating ENDOMETRIOSIS disease in a human patient, comprising: a) obtaining a sample of cells from the patient; b) obtaining a set of genotypes from the sample, wherein the set of genotypes comprises genotypes of one or more polymorphic loci from Tables 2-16; and c) comparing the set of genotypes of the sample with a set of genotypes associated with efficacy of the drug, wherein similarity between the set of genotypes of the sample and the set of genotypes associated with efficacy of the drug predicts the efficacy of the drug for treating ENDOMETRIOSIS disease in the patient.
82. The method of claim 81 , wherein the sample of cells is derived from a tissue selected from the group consisting of: ovarian cell, uterine cell, vaginal cell, skin, brain, nervous system, digestive system, respiratory system, scalp and reproductive system.
83. The method of claim 82, wherein the cells are selected from the group consisting of: ovarian cell, uterine cell, vaginal cell, hair cell, brain cell, muscle cell, neutrophil, dentric cell, T cell, mast cell, CD4+ lymphocyte, monocyte, macrophage, dendritic cell, and epithelial cell.
84. The method of claim 81 , wherein the sample is obtained via biopsy.
85. The method of claim 81 , wherein the set of genotypes from the sample comprises genotypes of at least two of the polymorphic loci listed in Tables 2-16.
86. The method of claim 81 wherein the set of genotypes from the sample is obtained by hybridization to allele-specific oligonucleotides complementary to the polymorphic loci from Tables 2-16, wherein said allele-specific oligonucleotides are contained on a microarray.
87. The method of claim 86, wherein the oligonucleotides comprise nucleic acid molecules at least 95% identical to SEQ ID from Tables 2-16.
88. The method of claim 81 wherein the set of genotypes from the sample is obtained by sequencing said polymorphic loci in said sample.
89. The method of claim 81 , wherein the drug is selected from the group consisting of symptom relievers and drugs for ENDOMETRIOSIS disease.
90. A method of treating ENDOMETRIOSIS disease in a patient in need thereof, comprising expressing in vivo at least one gene from Tables 2- 4 in an amount sufficient to treat the disease.
91. The method of claim 90, comprising: (a) administering to a patient a vector comprising a gene selected from Tables 2-4 that encodes the protein; and (b) allowing said protein to be expressed from said gene in said patient in an amount sufficient to treat the disease.
92. The method of claim 91 , wherein said vector is selected from the group consisting of an adenoviral vector, and a lentiviral vector.
93. The method of claim 91 , wherein said vector is administered by a route selected from the group consisting of: topical administration, intraocular administration, parenteral administration, intranasal administration, intratracheal administration, intrabronchial administration and subcutaneous administration.
94. The method of claim 91 , wherein said vector is a replication-defective viral vector.
95. The method of claim 91 , wherein said gene encodes a human protein.
96. A method of treating ENDOMETRIOSIS disease in a patient in need thereof, comprising administering an agent that regulates the expression, activity or physical state of at least one gene or its encoding RNA from Tables 2-4 in the patient.
97. The method of claim 96, wherein the encoded protein from said gene comprises an alteration.
98. The method of claim 96, wherein said gene comprises a mutation that modulates the expression of the encoded protein.
99. The method of claim 96, wherein said agent is selected from the group consisting of chemical compounds, oligonucleotides, peptides and antibodies.
100. The method of claim 99, wherein said agent is an antisense molecule or interfering RNA.
101. The method of claim 99, wherein said agent is an expression modulator.
102. The method of claim 101 , wherein said modulator is an activator.
103. The method of claim 101 , wherein said modulator is a repressor.
104. The method of claim 96, wherein said gene comprises a mutation that modifies at least one property or function of the encoded protein.
105. The method of claim 96, wherein the agent modulates at least one property or function of said gene.
106. A method of treating ENDOMETRIOSIS disease in a patient in need thereof, comprising administering an agent that regulates the expression, activity or physical state of at least one polypeptide encoded by a gene from Tables 2-4 in the patient.
107. The method of claim 106, wherein the encoded protein from said gene comprises an alteration, wherein said alteration is encoded by a polymorphic locus in said gene.
108. The method of claim 106, wherein said gene comprises an associated allele, a particular allele of a polymorphic locus, or the like that modulates the expression of the encoded protein.
109. The method of claim 106, wherein said agent is selected from the group consisting of chemical compounds, oligonucleotides, peptides and antibodies.
110. The method of claim 106, wherein said agent is an antisense molecule or interfering RNA.
111. The method of claim 106, wherein said agent is an expression modulator.
112. The method of claim 111 , wherein said modulator is an activator.
113. The method of claim 111 , wherein said modulator is a repressor.
114. The method of claim 106, wherein said gene comprises an associated allele, a particular allele of a polymorphic locus, or the like that modifies at least one property or function of the encoded protein.
115. A method for preventing the occurrence of ENDOMETRIOSIS disease in an individual in need thereof, comprising regulating the level of at least one gene from Tables 2-4 compared to a control.
116. The method of claim 115, wherein said level is regulated by regulating expression of at least one gene from Tables 2-4 by a binding agent, a receptor to said gene, a peptidomimetic, a fusion protein, a prodrug, an antibody or a ribozyme.
117. The method of claim 115, wherein said level is controlled by genetically altering the expression level of at least one gene from Tables 2-4, whereby the regulated level of said gene mimics the level in a healthy individual.
118. A method for identifying a gene that regulates drug response in ENDOMETRIOSIS disease, comprising: (a) obtaining a gene expression profile for at least one gene from Tables 2-4 in a resident tissue cell induced for a pro-inflammatory like state in the presence of the candidate drug; and (b) comparing the expression profile of said gene to a reference expression profile for said gene in a cell induced for the pro-inflammatory like state in the absence of the candidate drug, wherein genes whose expression relative to the reference expression profile is altered by the drug may identifies the gene as a gene that regulates drug response in ENDOMETRIOSIS disease.
119. A method for identifying an agent that alters the level of activity or expression of a polypeptide of Tables 2-4 for use in diagnostics, prognostics, prevention, treatment, or study of ENDOMETRIOSIS disease, comprising: (a) contacting a cell, cell lysate, or the polypeptide, with an agent to be tested; (b) assessing a level of activity or expression of the polypeptide; and (c) comparing the level of activity or expression of the polypeptide with a control sample in an absence of the agent, wherein if the level of activity or expression of the polypeptide in the presence of the agent differs by an amount that is statistically significant from the level in the absence of the agent then the agent alters the activity or expression of the polypeptide.
120. A kit for diagnosing susceptibility to ENDOMETRIOSIS disease in an individual comprising: primers for nucleic acid amplification of a region of at least one gene from Tables 2-4.
121. The kit of claim 120, wherein the primers comprise a segment of nucleic acids of length suitable for nucleic acid amplification of a target sequence, selected from the group consisting of: single nucleotide polymorphism from Tables 5-16, and combinations thereof.
122. A kit for assessing a patient's risk of having or developing ENDOMETRIOSIS disease, comprising: (a) detection means for detecting the differential expression, relative to a normal cell, of at least one gene shown in Table 4 or the gene product thereof; and (b) instructions for correlating the differential expression of said gene or gene product with a patient's risk of having or developing ENDOMETRIOSIS disease.
123. The kit of claim 122, wherein the detection means includes nucleic acid probes for detecting the level of mRNA of said genes.
124. A kit for assessing a patients risk of having or developing ENDOMETRIOSIS disease, comprising: (a) at least one means for amplifying or detecting a sequence of at least one gene in Tables 2-4, wherein the detection means includes nucleic acid probes or primers for detecting the presence or absence of mutations or changes to at least one sequence of Tables 2-4.
125. The kit of claim 124, wherein the detection means includes an immunoassay for detecting the level of at least one gene product from Tables 2-4.
126. A kit for assessing a patient's risk of having or developing ENDOMETRIOSIS disease, comprising: a) a detection means for detecting the genotype of at least one polymorphic locus shown in Tables 2-16; and b) instructions for correlating the genotype of said at least one polymorphic locus with a patient's risk of having or developing ENDOMETRIOSIS disease.
127. The kit of claim 126, wherein the detection means includes nucleic acid probes for detecting the genotype of said at least one polymorphic locus.
128. A diagnostic composition for diagnosing or detecting susceptibility to ENDOMETRIOSIS disease comprising a set of oligonucleotide probes that specifically hybridizes to at least two geneonic regions listed in Table 1.
129. The composition of claim 128, wherein said set of oligonucleotide probes specifically hybridize to sequences of at least two genes selected from the genes in Tables 2-4.
130. The composition of claim 128, wherein the oligonucleotide probes are detectably labeled with an agent selected from the group consisting of a fluorescent dye, a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate and an enzyme.
131. The composition of claim 130, wherein the oligonucleotide probes are labeled with different fluorescent compounds.
132. The composition of claim 128, wherein the set of oligonucleotide probes hybridizes in situ.
133. The composition of claim 128, wherein the set of oligonucleotide probes hybridizes at a gradually changing temperature.
134. The composition of claim 128, wherein the oligonucleotide probes are between 2 to 100 bases.
135. The composition of claim 128, wherein the oligonucleotide probes are between 3 to 50 bases.
136. The composition of claim 128, wherein the oligonucleotide probes are between 8 to 25 bases.
137. A method of assessing a patient's risk of having or developing ENDOMETRIOSIS disease, comprising: (a) determining the level of expression of at least one gene from Tables 2-4 or gene products thereof, and comparing the level of expression to a normal cell; and (b) assessing a patient's risk of having or developing ENDOMETRIOSIS disease by determining the correlation between the differential expression of said genes or gene products with known changes in expression of said genes measured in at least one patent suffering from ENDOMETRIOSIS disease.
138. A method of assessing a patient's risk of having or developing ENDOMETRIOSIS disease, comprising (a) determining a genotype for at least one polymorphic locus from Tables 2-16 in a patient; (b) comparing said genotype of (a) to a genotype for at least one polymorphic locus from Tables 2-16 that is associated with ENDOMETRIOSIS disease; and (c) assessing the patient's risk of having or developing ENDOMETRIOSIS disease, wherein said patient has a higher risk of having or developing ENDOMETRIOSIS disease if the genotype for at least one polymorphic locus from Tables 2-16 in said patient is the same as said genotype for at least one polymorphic locus from Tables 2-16 that is associated with ENDOMETRIOSIS disease.
139. A method for assaying the presence of a nucleic acid associated with resistance or susceptibility to ENDOMETRIOSIS disease in a sample, comprising: contacting said sample with a nucleic acid recited in claim 5 under stringent hybridization conditions; and detecting a presence of a hybridization complex.
140. A method for assaying the presence or amount of a polypeptide encoded by a gene of Tables 2-4 for use in diagnostics, prognostics, prevention, treatment, or study of ENDOMETRIOSIS disease, comprising: contacting a sample with an antibody that specifically binds to a gene of Tables 8, 9 or 10 under conditions appropriate for binding; and assessing the sample for the presence or amount of binding of the antibody to the polypeptide.
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