Expression monitoring of downstream genes in the BRCA1 pathway

Abstract

Analysis of the genes whose expression is affected by BRCA1 has identified a set of genes, each of which is up- or down-regulated by BRCA1. Each of these genes, alone or in groups, can be used to determine the mutational status of a BRCA1 gene, to determine whether a particular allelic variant affects BRCA1 function, to diagnose neoplasia, and to help identify candidate drugs which may be useful as anti-neoplastic agents.

1 . A method for detecting a BRCA1 gene functional mutation in target cells comprising the steps of: 
 detecting expression of a plurality of down-stream genes of BRCA1 in a sample of (a) target cells and (b) reference cells having a wild-type BRCA1 gene, the reference cells being otherwise substantially similar to the target cells, the down-stream genes being up- or down-regulated by the wild-type BRCA1 gene; and    comparing the expression of the down-stream genes in the target cells and the reference cells, wherein a difference in the expression between the target cells and reference cells suggests a BRCA1 functional mutation in the target cells.    
     
     
         2 . The method of    claim 1   , wherein said down-stream genes are transcriptionally regulated by said wild-type BRCA1 gene and the expression of said down-stream genes is detected by measuring amounts of transcripts of said down-stream genes in said reference and target cells.  
     
     
         3 . The method of    claim 2   , wherein said amounts of transcripts are measured with high density nucleic acid array.  
     
     
         4 . The method of    claim 1   , wherein said down-stream genes comprise at least one BRCA1 up-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, EtrIO1, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAKI), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, and Nucleotide binding protein.  
     
     
         5 . The method of    claim 1    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Pim-1, Pigf, and GF14Ω.  
     
     
         6 . The method of    claim 1    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Mki67a and prothymosin-α.  
     
     
         7 . The method of    claim 1    further comprising the step of: 
 indicating a loss of function mutation in the BRCA1 gene in the target cells if the expression of said BRCA1 up-regulated genes is at least two times less in said target cells than in said reference cells or if the expression of said BRCA1 down-regulated genes is at least two times more in said target cells than in said reference cells.  
 
     
     
         8 . An in-cell functional assay for a BRCA1 sequence alteration comprising the steps of: 
 detecting expression of a plurality of BRCA1 down-stream genes in a target sample from target cells having a BRCA1 sequence alteration and in a reference sample from reference cells having a wild-type BRCA1 gene, said reference cells being otherwise substantially similar to said target cells, said down-stream genes being up- or down-regulated by said wild-type BRCA1 gene; and    comparing said expression in said target sample to said expression in said reference sample, wherein a difference in the expression between said two samples suggests that said BRCA1 sequence alteration affects the biological function of BRCA1.    
     
     
         9 . The method of    claim 8   , wherein said down-stream genes are transcriptionally regulated by said wild-type BRCA1 gene and the expression of said down-stream genes is detected by measuring amounts of transcripts of said down-stream genes in said reference and target cells.  
     
     
         10 . The method of    claim 9   , wherein said amounts of transcripts are measured with a high density nucleic acid array.  
     
     
         11 . The method of    claim 8    wherein said down-stream genes comprise at least one BRCA1 up-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, and Nucleotide binding protein.  
     
     
         12 . The method of    claim 8    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Pim-1, Pigf, and GF14Ω.  
     
     
         13 . The method of    claim 8    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Mki67a and prothymosin-α.  
     
     
         14 . The method of    claim 8    further comprising the steps of: 
 indicating that said BRCA1 sequence alteration is a loss of wild-type function mutation if the expression of said BRCA1 up regulated genes is at least two times less in said target cells than in said reference cells or if the expression of said BRCA1 down regulated genes is at least two times more in said target cells than in said reference cells.  
 
     
     
         15 . A method for detecting a mutation in a target BRCA1 gene using a computer comprising: 
 inputting wild-type expression data of a plurality of down-stream genes in a wild-type sample containing a wild-type BRCA1 gene, said down-stream genes being transcriptionally regulated by said wild-type BRCA1 gene;    inputting target expression data of said plurality of down-stream genes in a target sample containing said target BRCA1 gene;    comparing the target and wild-type expresssion data to determine differences, wherein differences suggest a mutation in said target BRCA1 gene.    
     
     
         16 . The method of    claim 15    wherein said down-stream genes comprise at least one BRCA1 up-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, EtrIOl, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, and Nucleotide binding protein.  
     
     
         17 . The method of    claim 15    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Pim-1, Pigf, and GF 14Ω.  
     
     
         18 . The method of    claim 15    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Mki67a and prothymosin-α.  
     
     
         19 . The method of    claim 15    further comprising the step of: 
 indicating a mutation in said target BRCA1 gene if the expression of said BRCA1 up-regulated genes is at least two times less in said target cells than in said reference cells or if the expression of said BRCA1 down-regulated genes is at least two times more in said target cells than in said reference cells.  
 
     
     
         20 . A method of diagnosing neoplasia of a test cell comprising: 
 hybridizing a transcription indicator of a test cell to a set of nucleic acid probes, wherein the transcription indicator is selected from the group consisting of mRNA, cDNA and cRNA, wherein 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 BRCA1;    detecting amounts of transcription indicator which hybridize to each of said set of nucleic acid probes;    identifying a test cell as neoplastic if (1) hybridization of the transcription indicator of the test cell to a probe which is a BRCA1-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 BRCA1-repressed gene is higher than hybridization using a transcription indicator from a normal cell.    
     
     
         21 . The method of    claim 20    wherein the test cell is a breast cell.  
     
     
         22 . The method of    claim 20    wherein at least 4 of said probes comprise portions of genes which are BRCA1-activated or BRCA1-repressed.  
     
     
         23 . The method of    claim 20    wherein at least 10 of said probes comprise portions of genes which are BRCA1-activated or BRCA1-repressed.  
     
     
         24 . The method of    claim 20    wherein at least 15 of said probes comprise portions of genes which are BRCA1-activated or BRCA1-repressed.  
     
     
         25 . The method of    claim 20    wherein the nucleic acid probes are attached to a solid support.  
     
     
         26 . The method of    claim 20    wherein the nucleic acid probes are arranged in an array.  
     
     
         27 . The method of    claim 26    wherein the array comprises nucleic acid probes which are portions of at least 250 different genes.  
     
     
         28 . The method of    claim 26    wherein the array comprises nucleic acid probes which are portions of at least 6000 different genes.  
     
     
         29 . The method of    claim 20    wherein said down-stream genes comprise at least one BRCA1 up-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, and Nucleotide binding protein.  
     
     
         30 . The method of    claim 20    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Pim-1, Pigf, and GF14Ω.  
     
     
         31 . The method of    claim 20    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Mki67a and prothymosin-α.  
     
     
         32 . The method of    claim 20    further comprising the step of: 
 determining the sequence of BRCA1 genes in the test cell to determine the BRCA1 genotypic status of the cell.  
 
     
     
         33 . The method of    claim 20    wherein a test cell is identified as neoplastic if hybridization is at least 2-fold different between compared samples.  
     
     
         34 . The method of    claim 20    wherein a test cell is identified as neoplastic if hybridization is at least 3-fold different between compared samples.  
     
     
         35 . The method of    claim 20    wherein a test cell is identified as neoplastic if hybridization is at least 5-fold different between compared samples.  
     
     
         36 . The method of    claim 20    wherein a test cell is identified as neoplastic if hybridization is at least 10-fold different between compared samples.  
     
     
         37 . A method of identifying anti-cancer drugs, comprising the step of: 
 contacting a test compound with a human cell;    monitoring expression of a plurality of BRCA1-downstream genes;    identifying a test compound as a potential anti-cancer drug if it increases expression of a BRCA1 up-regulated gene or decreases expression of a BRCA1 down-regulated gene in the human cell.    
     
     
         38 . The method of    claim 37    wherein the cell is a breast cancer cell.  
     
     
         39 . The method of    claim 37    wherein the cell is a tumor cell.  
     
     
         40 . The method of    claim 37    wherein the cell contains no wild-type BRCA1 genes.  
     
     
         41 . The method of    claim 37   , wherein the expression of said BRCA1 upregulated and down-regulated genes is detected by measuring amounts of transcripts of said genes in said cell.  
     
     
         42 . The method of    claim 41   , wherein said amounts of transcripts are measured using a high density nucleic acid array.  
     
     
         43 . The method of    claim 37    wherein said down-stream genes comprise at least one BRCA1 up-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, and Nucleotide binding protein.  
     
     
         44 . The method of    claim 37    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Pim-1, Pigf, and GF14Ω.  
     
     
         45 . The method of    claim 37    wherein said down-stream genes comprise at least one BRCA1 down-regulated gene selected from the group consisting of Mki67a and prothymosin-α.  
     
     
         46 . A method for detecting a BRCA1 gene functional mutation in target cells comprising the steps of: 
 detecting expression of a down-stream gene of BRCA1 in a sample of (a) target cells, and (b) reference cells having a wild-type BRCA1 gene, said reference cells being otherwise substantially similar to said target cells, said down-stream gene being selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARDI, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α; and    comparing the expression of said down-stream gene in said target cells and said reference cells, wherein a difference in said expression between the target cells and reference cells suggests a BRCA1 functional mutation in the target cells.    
     
     
         47 . An in-cell functional assay for a BRCA1 sequence alteration comprising the steps of: 
 detecting expression of a down-stream BRCA1-regulated gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PMscl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α, in a target sample from target cells having a BRCA1 sequence alteration and in a reference sample from reference cells having a wild-type BRCA1 gene, said reference cells being otherwise substantially similar to said target cells; and    comparing said expression in said target sample to said expression in said reference sample, wherein a difference in the expression between said two samples suggests that said BRCA1 sequence alteration affects the biological function of BRCA1.    
     
     
         48 . A method for detecting a mutation in a target BRCA1 gene using a computer comprising: 
 inputting wild-type expression data of a down-stream gene in a wild-type sample containing a wild-type BRCA1 gene, said down-stream gene being transcriptionally regulated by said wild-type BRCA1 gene, said down-stream gene being selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α;    inputting target expression data of said down-stream gene in a target sample containing said target BRCA1 gene;    comparing the target and wild-type expresssion data to determine differences, wherein differences suggest a mutation in said target BRCA1 gene.    
     
     
         49 . A method of diagnosing neoplasia of a test cell comprising: 
 hybridizing a transcription indicator of a test cell to a nucleic acid probe, wherein the transcription indicator is selected from the group consisting of mRNA, cDNA and cRNA, wherein the nucleic acid probe comprises a portion of a gene which is activated by or repressed by BRCA1, said gene being selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α;    detecting amounts of transcription indicator which hybridize to said nucleic acid probe;    identifying a test cell as neoplastic if (1) hybridization of the transcription indicator of the test cell to a probe from a BRCA1-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 of a BRCA1-repressed gene is higher than hybridization using a transcription indicator from a normal cell.    
     
     
         50 . A method of identifying anti-cancer drugs, comprising the step of: 
 contacting a test compound with a human cell;    monitoring expression of a BRCA1-downstream gene selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD 1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK 1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α;    identifying a test compound as a potential anti-cancer drug if it increases expression of a BRCA1 up-regulated gene or decreases expression of a BRCA1 down-regulated gene in the human cell.    
     
     
         51 . A method of diagnosing neoplasia of a test cell comprising: 
 detecting expression in a test cell of a plurality of genes which are activated by or repressed by BRCA1;    identifying a test cell as neoplastic if (1) expression of at least one of said BRCA1-activated genes is lower in the test cell than expression in a normal cell, or (2) expression of at least one of said BRCA1-repressed genes is higher than expression in a normal cell.    
     
     
         52 . A method of diagnosing neoplasia of a test cell comprising: 
 detecting expression in a test cell of a gene which is activated by or repressed by BRCA1, wherein the gene is selected from the group consisting of gadd45, histone H4, EGR1, ATF3, Jun-B, Rev-Erb α, Etr101, A20, tristetraproline (TTP), Gem GTPase, Brl40, R12810, PM-scl75, ARD1, hepatic leukemia factor, amphiregulin, TR3, orphan receptor (NAK1), fibroblast activating protein-α, IL4 receptor α, R41997, H81220, KIAAA0027, Nucleotide binding protein, Pim-1, Pigf, GF14Ω, Mki67a and prothymosin-α;    identifying a test cell as neoplastic if (1) expression of a BRCA1-activated gene in the test cell is lower than expression of the gene in a normal cell, or (2) expression of a BRCA1-repressed gene in the test cell is higher than expression in a normal cell.    
     
     
         53 . The method of    claim 51    wherein expression is detected immunologically.  
     
     
         54 . The method of    claim 52    wherein expression is detected immunologically.