Methods and Compositions for the Diagnosis of Cancer Susceptibilities and Defective DNA Repair Mechanisms and Treatment Thereof

Abstract

Methods and compositions for the diagnosis of cancer susceptibilities, defective DNA repair mechanisms and treatments thereof are provided. Among sequences provided here, the FANCD2 gene has been identified, and probes and primers are provided for screening patients in genetic-based tests and for diagnosing Fanconi Anemia and cancer. The FANCD2 gene can be targeted in vivo for preparing experimental mouse models for use in screening new therapeutic agents for treating conditions involving defective DNA repair. The FANCD2 polypeptide has been sequenced and has been shown to exist in two isoforms identified as FANCD2-S and the monoubiquinated FANCD-L form. Antibodies including polyclonal and monoclonal antibodies have been prepared that distinguish the two isoforms and have been used in diagnostic tests to determine whether a subject has an intact Fanconi Anemia/BRCA pathway.

1 .- 41 . (canceled) 
     
     
         42 . An isolated monoubiquitinated polypeptide comprising an amino acid sequence selected from:
 (a) an amino acid sequence of SEQ ID NO: 1,   (b) an amino acid sequence of SEQ ID NO: 4, and   (c) an amino acid sequence encoded by a nucleotide sequence at least 90% identical to the nucleotide sequence of any one of SEQ ID NOs: 5-8 and 187-188,   
       wherein the monoubiquitinated polypeptide is ubiquitinated at lysine 561. 
     
     
         43 . A method for identifying a defect in the Fanconi Anemia pathway in a cell population from a subject, comprising:
 a) determining in vitro whether the amount of a monoubiquitinated polypeptide is reduced in the cell population compared with the amount of the monoubiquitinated polypeptide in a wild-type cell population; and   b) if the amount of the monoubiquitinated polypeptide is reduced, determining whether the amount of any of FANCA, FANCB, FANCC, FANCD1, FANCE, FANCF or FANCG protein is altered in the cell population compared with the wild-type cell population,   
       wherein the monoubiquitinated polypeptide is the monoubiquitinated polypeptide of  claim 42 . 
     
     
         44 . A method for identifying a therapeutic agent, comprising:
 i) selecting a cell population in which a monoubiquitinated polypeptide is made in a reduced amount;   ii) exposing the cell population to individual members of a library of candidate therapeutic molecules; and   iii) identifying individual member molecules that cause the amount of the monoubiquitinated polypeptide to increase or decrease,   
       wherein the monoubiquitinated polypeptide is the monoubiquitinated polypeptide of  claim 42 . 
     
     
         45 . The method of  claim 44 , wherein the cell population is an in vitro cell population or an in vivo cell population having a mutant FANCD2 gene. 
     
     
         46 . The method of  claim 44 , wherein a chemical carcinogen is added to the cell population in which the monoubiquitinated polypeptide is made in reduced amounts to determine if any individual member molecules can cause the amount of the monoubiquitinated polypeptide to increase. 
     
     
         47 . A method for identifying a cell having a mutant FANCD2 allele from a subject, comprising:
 (a) determining the amount of a monoubiquitinated polypeptide in the cell; and   (b) comparing the amount in (a) with the amount of the monoubiquitinated polypeptide in a wild-type cell,   
       wherein the monoubiquitinated polypeptide is the monoubiquitinated polypeptide of  claim 42 , and wherein a reduction of the amount in (a) compared with the amount in (b) indicates that the cell has a mutant FANCD2 allele. 
     
     
         48 . The method of  claim 47 , wherein the mutant FANCD2 allele is a somatic allele in a tumor type and identifying the cell having the mutant FANCD2 allele is diagnostic for the tumor type. 
     
     
         49 . The method of  claim 47 , wherein the mutant FANCD2 allele is a germline allele. 
     
     
         50 . The method of  claim 47 , wherein identification of the cell having a mutant FANCD2 allele is indicative of a predisposition for a cancer in the subject or an increased risk of the subject bearing an offspring with Fanconi Anemia. 
     
     
         51 . A method for determining a therapeutic protocol for a subject having a cancer, comprising determining the amount of a monoubiquitinated polypeptide in cancer cells from the subject, comprising:
 (a) determining if the amount of the monoubiquitinated polypeptide in the cancer cells is reduced relative to the amount present in noncancerous cells from the subject;   (b) if a reduction is detected in (a), determining whether the reduction is a result of the same genetic defect present in both the cancer cells and the noncancerous cells; and   (c) if (b) is positive, reducing the use of the therapeutic protocol that causes increased DNA damage; or   (d) if (b) is negative, increasing the use of the therapeutic protocol that causes increased DNA damage,   
       wherein the monoubiquitinated polypeptide is the monoubiquitinated polypeptide of  claim 42 . 
     
     
         52 . A method for treating a FA pathway defect in a cell target by administering the monoubiquitinated polypeptide of  claim 42 . 
     
     
         53 . A method for detecting a FA pathway defect, comprising:
 (a) obtaining a cell sample from a subject;   (b) exposing the cell sample to a DNA damaging agent; and   (c) detecting whether the amount of the monoubiquitinated polypeptide of  claim 42  is upregulated,   
       wherein the absence of upregulation is indicative of a FA pathway defect. 
     
     
         54 . The method of  claim 53 , wherein the amount of noubiquitinated polypeptide is measured by an analysis technique selected from immunohistochemistry and Western blots.