Claims
- 1. An isolated nucleic acid molecule, comprising: a polynucleotide selected from
(a) a nucleotide sequence encoding a polypeptide having an aminoacid sequence as shown in SEQ ID NO: 4 (b) a nucleotide sequence at least 90% identical to the nucleotide sequence of (a); (c) a nucleotide sequence complementary to the nucleotide sequence of (b); (d) a nucleotide sequence at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 5-8, 187-188; and (e) a nucleotide sequence complementary to the nucleotide sequence of (d).
- 2. An isolated nucleic acid molecule according to claim 1, wherein the polynucleotide is a DNA molecule.
- 3. An isolated nucleic acid molecule of claim 2, wherein the polynucleotide is cDNA.
- 4. An isolated nucleic acid molecule according to claim 1, wherein the polynucleotide is an RNA molecule.
- 5. An isolated nucleic acid molecule consisting essentially of a nucleotide sequence encoding a polypeptide having an amino acid sequence sufficiently similar to that of SEQ ID NO: 4 to retain the biological property of conversion from a short form to a long form of FANCD2 in the nucleus of a cell for facilitating DNA repair.
- 6. An isolated nucleic acid molecule consisting essentially of a polynucleotide having a nucleotide sequence at least 90% identical to SEQ ID NO: 9-191 or complementary to a nucleotide sequence that is at least 90% identical to SEQ ID NO: 9-191.
- 7. An isolated nucleic acid molecule according to claim 6, wherein the sequence is an intron/exon sequence selected from SEQ ID NO: 9-94 disclosed in Table 6.
- 8. An isolated nucleic acid molecule according to claim 6, wherein the sequence is a PCR primer selected from SEQ ID NO: 115-186 disclosed in Table 7.
- 9. A method for making a recombinant vector comprising inserting the isolated nucleic acid molecule of claim I into a vector.
- 10. A recombinant vector produced by the method of claim 9.
- 11. A method of making a recombinant host cell comprising: introducing the recombinant vector of claim 10 into a host cell.
- 12. A recombinant host cell produced by the method of claim 11.
- 13. A method of making an FA—D2 cell line, comprising:
(a) obtaining cells from a subject having a biallelic mutation in a complementation group associated with FA—D2; and (b) infecting the cells with a transforming virus to make the FA—D2 cell line.
- 14. A method according to claim 13, wherein the cells are selected from fibroblasts and lymphocytes.
- 15. A method according to claim 13, wherein the transforming virus is selected from Epstein Barr virus and retrovirus.
- 16. A method according to claim 13, further comprising: characterizing the FA—D2 cell line by determining the presence of a defective FANDC2.
- 17. A method according to claim 16, wherein characterizing the FA—D2 cell line further comprises:
performing a diagnostic assay on the cell line, the diagnostic assay selected from (i) a Western blot or nuclear immunofluorescence using an antibody specific for FANCD2 and (ii) a DNA hybridization assay.
- 18. A recombinant method for producing a polypeptide, comprising: culturing a recombinant host cell wherein the host cell comprises the isolated nucleic acid molecule of claim 1.
- 19. An isolated polypeptide, comprising an aminoacid sequence selected from
(a) SEQ ID NO: 4; (b) an aminoacid sequence at least 90% identical to (a); (c) an aminoacid sequence which is encoded by a polynucleotide having a nucleotide sequence which is at least 90% identical to at least one of SEQ ID NO: 5-8, 187-188; and (d) a polypeptide fragment of (a) -(d) wherein the fragment is at least 50 aminoacids in length.
- 20. An isolated polypeptide according to claim 19, encoded by a DNA having a mutation selected from nt 376 A to G, nt 3707 G to A, nt9O4C to T and nt 958C to T.
- 21. An isolated polypeptide according to claim 19, the polypeptide characterized by a polymorphism in DNA encoding the polypeptide, the polymorphism being selected from nt 1122A to G, nt 1440T to C, nt1509C to T, nt2141C to T, nt2259T to C, nt4098T to G, nt4453G to A.
- 22. An isolated polypeptide according to claim 19, the polypeptide characterized by a mutation at aminoacid 222 or aminoacid 561.
- 23. An antibody preparation having a binding specificity for a FAN0CD2 protein.
- 24. An antibody preparation according to claim 23, further comprising: monoclonal antibodies.
- 25. An antibody preparation according to claim 23, further comprising: polyclonal antibodies.
- 26. An antibody preparation according to claim 23, wherein the FANCD2 protein is FANCD2-S.
- 27. An antibody preparation according to claim 23, wherein the FANCD2 protein is FANCD2-L.
- 28. A diagnostic method for measuring FANCD2 isoforms in a biological sample, the method comprising:
(a) exposing the sample to a first antibody for forming a first complex with FANCD2-L and optionally a second antibody for forming a second complex with FANCD2-S ; and (b) detecting with a marker, the amount of the first complex and the second complex in the sample.
- 29. A diagnostic method according to claim 28, wherein the sample comprises intact cells.
- 30. A diagnostic method according to claim 28, wherein the sample comprises lysed cells in a lysate.
- 31. A diagnostic method according to claim 28, wherein the biological sample is from a human subject with a susceptibility to cancer or having the initial stages of cancer.
- 32. A diagnostic method according to claim 31, wherein the biological sample is from a cancer in a human subject, wherein the cancer is selected from melanoma, leukemia, astocytoma, glioblastoma, lymphoma, glioma, Hodgkins lymphoma, chronic lymphocyte leukemia and cancer of the pancreas, breast, thyroid, ovary, uterus, testis, pituitary, kidney, stomach, esophagus and rectum.
- 33. A diagnostic method according to claim 28, wherein the biological sample is from a human fetus.
- 34. A diagnostic method according to claim 28, wherein the biological sample is from an adult human.
- 35. A diagnostic method according to claim 28, wherein the biological sample is selected from: a blood sample, a biopsy sample of tissue from the subject and a cell line.
- 36. A diagnostic method according to claim 28, wherein the biological sample is derived from heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon, peripheral blood and lymphocytes.
- 37. A diagnostic method according to claim 28, wherein the marker is a fluorescent marker, the fluorescent marker optionally conjugated to the FANCD2-L antibody.
- 38. A diagnostic method according to claim 28, wherein the marker is a chemiluminescent marker, the chemiluminescent marker optionally conjugated to the FANCD2-L antibody.
- 39. A diagnostic method according to claim 28, further comprising: binding the first and the second complex to a third antibody conjugated to a substrate.
- 40. A diagnostic method according to claim 30, wherein the lysate is subjected to a separation procedure to separate FANCD2 isoforms and the seperated isoforms are identified by determining binding to the first or the second FANCD2 antibody.
- 41. A diagnostic test for identifying a defect in the Fanconi Anemia pathway in a cell population from a subject, comprising:
selecting an antibody to FANCD2 protein and determining whether the amount of an FAND2-L isoform is reduced in the cell population compared with amounts, in a wild type cell population; such that if the amount of the FANCD2-L protein is reduced, then determining whether an amount of any of FANCA, FANCB, FANCC, FANCD1, FANCE, FANCF or FANCG protein is altered in the cell population compared with the wild type so as to identify the defect in the Fanconi Anemia pathway in the cell population.
- 42. A diagnostic test according to claim 41, wherein determining the amount of an isoform relies on a separation of the FANCD2-L and FANCD2-S isoforms.
- 43. A diagnostic test according to claim 41, wherein the separation is achieved by gel electrophoresis.
- 44. A diagnostic test according to claim 41, wherein the separation is achieved by a migration binding banded test strip.
- 45. A screening assay for identifying a therapeutic agent, comprising:
selecting a cell population in which FAND2-L is made in reduced amounts; exposing the cell population to individual members of a library of candidate therapeutic molecules; and identifying those individual member molecules that cause the amount of FANCD2-L to be increased in the cell population.
- 46. A screening assay according to claim 45, wherein the cell population is an in vitro cell population.
- 47. A screening assay according to claim 45, wherein the cell population is an in vivo cell population, the in vivo population being within an experimental animal, the experimental animal having a mutant FANCD2 gene.
- 48. A screening assay according to claim 45, wherein the experimental animal is a knock-out mouse in which the mouse FAND2 gene has been replaced by a human mutant FANCD2 gene.
- 49. A screening assay according to claim 45, wherein a chemical carcinogen is added to the cell population in which FANCD2 is made in reduced amounts, to determine if any member molecules can cause the amount of FANCD2-L to be increased so as to protect the cells form the harmful effects of the chemical carcinogen.
- 50. An experimental animal model in which the animal FANCD2 gene has been removed and optionally replaced by a nucleic acid molecule of claim 1.
- 51. A method for identifying in a cell sample from a subject, a mutant FANCD2 nucleotide sequence in a suspected mutant FANCD2 allele which comprises comparing the nucleotide sequence of the suspected mutant FANCD2 allele with the wild type FANCD2 nucleotide sequence wherein a difference between the suspected mutant and the wild type sequence identifies a mutant FANCD2 nucleotide sequence in the cell sample.
- 52. A method according to claim 51, wherein the suspected mutant allele is a germline allele.
- 53. A method according to claim 51, wherein identification of a mutant FANCD2 nucleotide sequence is diagnostic for a predisposition for a cancer in the subject.
- 54. A method according to claim 51, wherein identification of a mutant FANCD2 nucleotide sequence is diagnostic for an increased risk of the subject bearing an offspring with Fanconi Anemia.
- 55. A method according to claim 51, wherein the suspected mutant allele is a somatic allele in a tumor type and identifying a mutant FANCD2 nucleotide sequence is diagnostic for the tumor type.
- 56. A method according to claim 51, wherein the nucleotide sequence of the wild type and the suspected mutant FANCD2 nucleotide sequence is selected from a gene, a mRNA and a cDNA made from a mRNA.
- 57. A method according to claim 51, wherein comparing the polynucleotide sequence of the suspected mutant FANCD2 allele with the wild type FANCD2 polynucleotide sequence, further comprises: selecting a FANCD2 probe which specifically hybridizes to the mutant FANCD2 nucleotide sequence, and detecting the presence of the mutant sequence by hybridization with the probe.
- 58. A method according to claim 51, wherein comparing the polynucleotide sequence of the suspected mutant FANCD2 allele with the wild type FANCD2 polynucleotide sequence, further comprises amplifying all or part of the FANCD2 gene using a set of primers specific for wild type FANCD2 DNA to produce amplified FANCD2 DNA and sequencing the FANCD2 DNA so as to identify the mutant sequence.
- 59. A method according to claim 51, wherein the mutant FANCD2 nucleotide sequence is a germline alteration in the FANCD2 allele of the human subject, the alteration selected from the alterations set forth in Table 3.
- 60. A method according to claim 51, wherein the mutant FANCD2 nucleotide sequence is a somatic alteration in the FANCD2 allele of the human subject, the alteration selected from the alterations set forth in Table 3.
- 61. A method for diagnosing a susceptibility to cancer in a subject which comprises comparing the germline sequence of the FANCD2 gene or the sequence of its mRNA in a tissue sample from the subject with the germline sequence of the FANCD2 gene or the sequence of its mRNA wherein an alteration in the germline sequence of the FANCD2 gene or the sequence of its mRNA of the subject indicates the susceptibility to the cancer.
- 62. A method according to claim 61, wherein an alteration is detected in a regulatory region of the FANCD2 gene.
- 63. A method according to claim 61, wherein the detection in the alteration in the germline sequence is determined by an assay selected from the group consisting of (a) observing shifts in electrophoretic mobility of single-stranded DNA on non-denaturing polyacrylamide gels, (b) hybridizing a FANCD2 gene probe to genomic DNA isolated from the tissue sample, (c) hybridizing an allele-specific probe to genomic DNA of the tissue sample, (d) amplifying all or part of the FANCD2 gene from the tissue sample to produce an amplified sequence and sequencing the amplified sequence, (e) amplifying all or part of the FANCD2 gene from the tissue sample using primers for a specific FANCD2 mutant allele, (f) molecular cloning all or part of the FANCD2 gene from the tissue sample to produce a cloned sequence and sequencing the cloned sequence, (g) identifying a mismatch between (i) a FANCD2 gene or a FANCD2 mRNA isolated from the tissue sample, and (ii) a nucleic acid probe complementary to the human wild-type FANCD2 gene sequence, when molecules (i) and (ii) are hybridized to each other to form a duplex, (h) amplification of FANCD2 gene sequences in the tissue sample and hybridization of the amplified sequences to nucleic acid probes which comprise wild-type FANCD2 gene sequences, (I) amplification of FANCD2 gene sequences in the tissue sample and hybridization of the amplified sequences to nucleic acid probes which comprise mutant FANCD2 gene sequences, (j) screening for a deletion mutation in the tissue sample, (k) screening for a point mutation in the tissue sample, (1) screening for an insertion mutation in the tissue sample, (m) in situ hybridization of the FANCD2 gene of the tissue sample with nucleic acid probes which comprise the FANCD2 gene.
- 64. A method of diagnosing a susceptibility for cancer in a subject, comprising:
(a) accessing genetic material from the subject so as to determine defective DNA repair; (b) determining the presence of mutations in a set of genes, the set comprising FAND2 and at least one of FANCA, FANCB, FANCC, FANCD1, FANCDE, FANDF, FANDG, BRACAL and ATM; and (c) diagnosing susceptibility for cancer from the presence of mutations in the set of genes.
- 65. A method for detecting a mutation in a neoplastic lesion at the FANCD2 gene in a human subject which comprises:
comparing the sequence of the FANCD2 gene or the sequence of its mRNA in a tissue sample from a lesion of the subject with the sequence of the wild-type FANCD2 gene or the sequence of its mRNA, wherein an alteration in the sequence of the FANCD2 gene or the sequence of its MRNA of the subject indicates a mutation at the FANCD2 gene of the neoplastic lesion.
- 66. A method according to claim 65, further comprising: determining a therapeutic protocol for treating the neoplastic lesion according to the mutation at the FANCD2 gene of the neoplastic lesion.
- 67. A method for confirming the lack of a FANCD2 mutation in a neoplastic lesion from a human subject which comprises comparing the sequence of the FANCD2 gene or the sequence of its mRNA in a tissue sample from a lesion of said subject with the sequence of the wild-type FANCD2 gene or the sequence of its RNA, wherein the presence of the wild-type sequence in the tissue sample indicates the lack of a mutation at the FANCD2 gene.
- 68. A method for determining a therapeutic protocol for a subject having a cancer, comprising:
(a) determining if a deficiency in FANCD2-L occurs in a cell sample from the subject by measuring FANCD2 isoforms according to claim 25;(b) if a deficiency is detected in (a) , then determining whether the deficiency is a result of genetic defect in non-cancer cells; and (c) if (b) is positive, reducing the use of a therapeutic protocol that causes increased DNA damage so as to protect normal tissue in the subject and if (b) is negative, and the deficiency is contained within a genetic defect in cancer cells only, then increasing the use of a therapeutic protocol that causes increased DNA damage so as to adversely affect the cancer cells.
- 69. A method of treating a FA pathway defect in a cell target, comprising: administering an effective amount of FANCD2 protein or an exogenous nucleic acid to the target.
- 70. A method according to claim 69, wherein the FA pathway defect is a defective FANCD2 gene and the exogenous nucleic acid vector further comprises introducing a vector according to claim 10.
- 71. A method according to claim 69, wherein the vector is selected from a mutant herpesvirus, a E1/E4 deleted recombinant adenovirus, a mutant retrovirus, the viral vector being defective in respect of a viral gene essential for production of infectious new virus particles.
- 72. A method according to claim 69, wherein the vector is contained in a lipid micelle.
- 73. A method for treating a patient with a defective FANCD2 gene, comprising:
providing a polypeptide described in SEQ ID No: 4, for functionally correcting a defect arising from a condition arising from the defective FANCD2 gene.
- 74. A cell based assay for detecting a FA pathway defect, comprising:
(a) obtaining a cell sample from a subject; (b) exposing the cell sample to DNA damaging agents; and (c) detecting whether FANCD2-L is upregulated, the absence of upregulation being indicative of the FA pathway defect.
- 75. A cell based assay according to claim 74, wherein amounts of FANCD2 are measured by an analysis technique selected from: immunoblotting for detecting nuclear foci; Western blots to detect amounts of FANCD2 isoforms and quantifying mRNA by hybridising with DNA probes.
- 76. A kit for use in detecting a cancer cell in a biological sample, comprising:
(a) a primer pair which binds under high stringency conditions to a sequence in the FANCD2 gene, the primer pair being selected to specifically amplify an altered nucleic acid sequence described in Table 7; and (b) containers for each of the primers.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application gains priority from provisional application 60/245,756 filed Nov. 3, 2000, the application being incorporated by reference herein.
Government Interests
[0002] The work described herein was supported by the National Institute of Health, NIH Grant vNo. Health grants RO1HL52725-04, RO1 DK43889-09, 1PO1HL48546, and PO1HL54785-04. The US Government has certain rights to the claimed invention
Provisional Applications (1)
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Number |
Date |
Country |
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60245756 |
Nov 2000 |
US |