Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-24
2001-01-23
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S440000, C536S023100
Reexamination Certificate
active
06177248
ABSTRACT:
BACKGROUND OF THE INVENTION
Many biological functions are accomplished by altering the expression of various genes through transcriptional (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle regulation, cell differentiation and cell death, are often characterized by the variations in the expression levels of groups of genes.
Gene expression is also associated with pathogenesis. For example, the lack of sufficient expression of functional tumor suppressor genes and/or the over expression of oncogene/protooncogenes could lead to tumorigenesis (Marshall,
Cell,
64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991), incorporated herein by reference for all purposes). Thus, changes in the expression levels of particular genes (e.g. oncogenes or tumor suppressors) serve as signposts for the presence and progression of various diseases. There is a need in the art for discovering which genes are affected by particular tumor suppressors and oncogenes, so that they can be used diagnostically as well as in the search for new therapeutics.
SUMMARY OF THE INVENTION
It is an object of the invention to provide methods for identifying a functional mutation in a WT1 gene.
It is another object of the invention to provide methods for identifying a EWS-WT1 gene fusion.
It is still another object of the invention to provide an in-cell assay to test the biological effect of a WT1 mutation.
It is yet another object of the invention to provide methods to detect a WT1 mutation or a EWS-WT1 fusion using a computer.
It is a further object of the invention to provide methods to diagnose neoplasia.
It is another object of the invention to provide methods for identifying drugs useful for treating neoplasias.
These and other objects of the invention are provided by one or more of the following embodiments. In one embodiment a method is provided for detecting a WT1 gene functional mutation in target cells. Expression of at least three down-stream genes of WT1 is detected in a sample of (a) target cells, and (b) reference cells having a wild-type WT1 gene. The reference cells are otherwise substantial similar to the target cells. The down-stream genes are up- or down-regulated by the wild-type WT1 gene. The expression of the down-stream genes in the target cells and the reference cells is compared; a difference in the expression between the target cells and reference cells suggests a WT1 functional mutation in the target cells.
According to yet another embodiment a method is provided for detecting a EWS-WT1 gene fusion in target cells. Expression of one or more down-stream genes of an EWS-WT1 fusion is detected in a sample of (a) target cells, and (b) reference cells having a wild-type EWS and WT1 gene. The reference cells are otherwise substantially similar to the target cells. The down-stream genes are up- or down-regulated by the wild-type EWS and WT1 gene. The expression of the down-stream genes in the target cells and the reference cells is compared. A difference in the expression between the target cells and reference cells suggests a EWS-WT1 fusion in the target cells.
Another aspect of the invention is an in-cell functional assay for a WT1 sequence alteration. The expression is detected of at least three down-stream genes in a target sample from target cells having a WT1 sequence alteration and in a reference sample from reference cells having a wild-type WT1 gene. The reference cells are otherwise substantially similar to the target cells. The down-stream genes are up- or down-regulated by the wild-type WT1 gene. The expression in the target sample is compared to the expression in the reference sample. A difference in the expression between the two samples suggests that the WT1 sequence alteration affects the biological function of WT1.
According to another aspect of the invention a method is provided for detecting a mutation in a target WT1 gene using a computer. Wild-type expression data of at least three down-stream genes in a wild-type sample containing a wild-type WT1 gene is input into a computer. The down-stream genes are transcriptionally regulated by the wild-type WT1 gene. Target expression data of the plurality of down-stream genes in a target sample containing the target WT1 gene is also input into a computer. The target and wild-type expression data are compared to determine differences. Differences suggest a mutation in the target WT1 gene.
In still another embodiment a method is provided for detecting a translocation fusing EWS and WT1 genes using a computer. Wild-type expression data of a plurality of down-stream genes in a wild-type sample containing wild-type EWS and WT1 genes is input into a computer. The down-stream genes are transcriptionally regulated by a EWS-WT 1 fusion protein. Target expression data of the plurality of down-stream genes in a target sample which is being tested for the presence of a EWS-WT1 fusion protein is also input into a computer. The target and wild-type expression data are compared to determine differences. Such differences suggest a translocation fusing the EWS and WT1 genes.
Another embodiment of the invention provides a method of diagnosing neoplasia of a test cell. A transcription indicator of a test cell is hybridized to a set of nucleic acid probes. The transcription indicator is selected from the group consisting of mRNA, cDNA and cRNA The set of nucleic acid probes comprises a plurality of nucleic acid molecules each of which is a portion of a gene which is activated by or repressed by WT1 selected from the group consisting of: natural killer cells protein 4 precursor (M59807), heat shock protein HSP70B (X51758), 90 K product (H17969), heat shock 70 kd protein 1 (T66307), procollagen alpha 1 (T51558), cysteine protease inhibitor from radiated keratinocytes (X05978), brain natriuretic protein (BNP) (M31766), leukemia virus receptor 1 (GLVR1) (20859), type 1 cytoskeletal 17 keratin (R71870), purine nucleoside phosphorylase (T47964), adrenomedullin (D14874), gravin (M96322), jun-B (X51345), elongation factor 1 alpha-2 (X79490), homeotic gene regulator (R16977), clone 9112 (X57348), interferon gamma treatment inducible mRNA (M26683), transmembrane receptor protein (Z17227) and mitochondrial phosphate carrier protein (R49231). Amounts of transcription indicator which hybridize to each of the set of nucleic acid probes are detected. A test cell is identified as neoplastic if (1) hybridization of the transcription indicator of the test cell to a probe which is a WT1-activated gene is lower than hybridization using a transcription indicator from a normal cell, or (2) hybridization of the transcription indicator of the test cell to a probe which a WT1-repressed gene is higher than hybridization using a transcription indicator from a normal cell.
A further aspect of the invention is provided by a method of identifying anti-cancer drugs. A test compound is contacted with a human cell. The effect of the test compound on the expression by the cell of at least one WT1 up- or down-regulated gene is determined. The regulated gene is selected from the group consisting of: natural killer cells protein 4 precursor (M59807), heat shock protein HSP70B (51758), 90 K product (H17969), heat shock 70 kd protein 1 (T66307), procollagen alpha 1 (T51558), cysteine protease inhibitor from radiated keratinocytes (X05978), brain natriuretic protein (BNP) (M31766), leukemia virus receptor 1 (GLVR1) (L20859), type 1 cytoskeletal 17 keratin, purine nucleoside phosphorylase (T47964), adrenomedullin (D14874), gravin (M96322), jun-B (X51345), elongation factor 1 alpha-2 (X79490), homeotic gene regulator (R16977), clone 9112 (X57348), interferon gamma treatment inducible mRNA (M26683), transmembrane receptor protein (Z17227) and mitochondrial phosphate carrier protein (R49231). A test compound is identified as a potential anti-cancer drug if it increases expression of at least one WT1 up-regulated gene or decreases expression of a WT1 down-regu
Haber Daniel
Lee Sean
Oliner Jonathan
Truong Vivi
Affymetrix Inc.
Banner & Witcoff , Ltd.
Brusca John S.
Siu Stephen
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