Method for detecting nucleosomes containing histone variants

Abstract

The invention relates to a method for detecting and measuring the presence of mono-nucleosomes and oligo-nucleosomes and nucleosomes that contain particular histone variants and the use of such measurements for the detection and diagnosis of disease. The invention also relates to a method of identifying histone variant biomarkers for the detection and diagnosis of disease and to biomarkers identified by said method.

It is claimed: 
     
         1 . A cell free nucleosome comprising a histone variant or histone isoform for use as a biomarker for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis, chronic obstructive pulmonary disorder, Crohn’s disease and rheumatoid arthritis. 
     
     
         2 . The nucleosome for use as defined in  claim 1  wherein the nucleosome is a mononucleosome or an oligonucleosome. 
     
     
         3 . The nucleosome for use as defined in  claim 1  or  claim 2  wherein the cell free nucleosome comprising a histone variant or histone isoform is measured in a blood sample. 
     
     
         4 . The nucleosome for use as defined in any one of  claims 1 to 3 , wherein the cancer is a cancer of the bladder, breast, colon, cervix, esophagus, kidney, large intestine, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach. 
     
     
         5 . The nucleosome for use as defined in  claim 4 , wherein the cancer is a cancer of the colon, lung, oral cavity or pancreas. 
     
     
         6 . A method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample which comprises the steps of: 
 (i) contacting the sample with a binding agent which binds to the histone variant or histone isoform;   (ii) detecting or quantifying the binding of said binding agent to the histone variant or histone isoform in the sample; and   (iii) using the presence or degree of such binding as a measure of the presence of the histone variant or histone isoform or nucleosomes containing the histone variant or histone isoform in the sample.   
     
     
         7 . A method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample which comprises the steps of:
 (i) contacting the sample with a first binding agent which binds to nucleosomes;   (ii) contacting the nucleosomes or sample with a second binding agent which binds to the histone variant or histone isoform;   (iii) detecting or quantifying the binding of said second binding agent to histone variant or histone isoform in the sample; and   (iv) using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.   
     
     
         8 . A method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample which comprises the steps of:
 (i) contacting the sample with a first binding agent which binds to the histone variant or histone isoform;   (ii) contacting the nucleosomes or sample with a second binding agent which binds to nucleosomes;   (iii) detecting or quantifying the binding of said second binding agent to nucleosomes in the sample; and   (iv) using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.   
     
     
         9 . The method as defined in any one of  claims 6-8  wherein the histone isoform is a common histone isoform that occurs in all or most nucleosomes to test for nucleosomes per se. 
     
     
         10 . The method as defined in any one of  claims 6-9  wherein the binding agent used to bind a histone is targeted to bind a region of the said histone that is common to all or most histone variants or isoforms of the said histone moiety and occurs in all or most nucleosomes to test for nucleosomes per se. 
     
     
         11 . A method as defined in any one of  claims 6 to 10  wherein the binding agent is an antibody. 
     
     
         12 . A method according to any one of  claims 6 to 11  wherein the sample is a biological fluid. 
     
     
         13 . A method according to any one of  claims 6 to 12  wherein the sample is blood, serum or plasma. 
     
     
         14 . A method for detecting the presence of a nucleosome containing a histone variant or histone isoform as defined in any one of  claims 6 to 13 , in a cell which comprises the steps of:
 (i) isolating chromatin from a cell;   (ii) digesting, sonicating or otherwise breaking down the chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and   (iii) detecting or measuring the presence of the histone variant or histone isoform in the said nucleosomes according to a method of any one of  claims 6 to 13 .   
     
     
         15 . A method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a blood, serum or plasma sample which comprises the steps of:
 (i) removing, releasing or extracting the histone variant or isoform from the nucleosome complex to produce a free histone variant or isoform moiety   (ii) detecting or quantifying the free histone variant or isoform in the sample; and   (iii) using the presence or amount of free histone variant or isoform as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.   
     
     
         16 . A method for detecting or diagnosing a disease status in an animal or a human subject which comprises the steps of:
 (i) detecting or measuring nucleosomes containing a histone variant or histone isoform in a body fluid of a subject; and   (ii) using the nucleosome associated histone variant or histone isoform level detected to identify the disease status of the subject.   
     
     
         17 . A method for assessment of an animal or a human subject for suitability for a medical treatment which comprises the steps of:
 (i) detecting or measuring nucleosomes containing a histone variant or histone isoform in a body fluid of the subject; and   (ii) using the nucleosome associated histone variant or histone isoform level detected as a parameter for selection of a suitable treatment for the subject.   
     
     
         18 . A method for monitoring a treatment of an animal or a human subject which comprises the steps of:
 (i) detecting or measuring nucleosomes containing a histone variant or histone isoform in a body fluid of the subject;   (ii) repeating the detection or measurement of nucleosomes containing a histone variant or histone isoform in a body fluid of the subject on one or more occasions; and   (iii) using any changes in the nucleosome associated histone variant or histone isoform level detected as a parameter for any changes in the condition of the subject.   
     
     
         19 . A method according to any one of  claims 16-18  wherein the nucleosome associated histone variant or histone isoform is detected or measured as one of a panel of measurements. 
     
     
         20 . A method for identifying a histone variant or histone isoform biomarker for 
 detecting or diagnosing a disease status in an animal or a human subject which comprises the steps of:   (i) detecting or measuring nucleosomes containing the histone variant or histone isoform in a body fluid of the subject;   (ii) detecting or measuring nucleosomes containing the histone variant or   histone isoform in a body fluid of a healthy subject or a control subject; and   (iii) using the difference between the levels detected in diseased and control subjects to identify whether a histone variant or histone isoform is useful as a biomarker for the disease status.   
     
     
         21 . A biomarker identified by the method as defined in  claim 20 . 
     
     
         22 . A kit for the detection of a nucleosome associated histone variant or histone isoform which comprises a ligand or binder specific for the histone variant or histone isoform or component part thereof, or a structural/shape mimic of the histone variant or histone isoform or component part thereof, together with instructions for use of the kit in accordance with any one of the methods defined in  claims 6 to 20 .