DETECTION OF METHYLATED CpG RICH SEQUENCES DIAGNOSTIC FOR MALIGNANT CELLS

Abstract

The present invention provides methods for determining the methylation status of CpG-containing dinucleotides on a genome-wide scale using infrequent cleaving, methylation sensitive restriction endonucleases and two-dimensional gel electrophoretic display of the resulting DNA fragments. Such methods can be used to diagnose cancer, classify tumors and provide prognoses for cancer patients. The present invention also provides isolated polynucleotides and oligonucleotides comprising CpG dinucleotides that are differentially methylated in malignant cells as compared to normal, non-malignant cells. Such polynucleotides and oligonucleotides are useful for diagnosis of cancer. The present invention also provides methods for identifying new DNA clones within a library that contain specific CpG dinucleotides that are differentially methylated in cancer cells as compared to normal cells.

1 . A method for characterizing whether cells isolated from a subject suspected as malignant or non-malignant, comprising: 
 a) digesting DNA isolated from the cells with a methylation-sensitive restriction enzyme to provide a set of test restriction fragments;    b) digesting DNA isolated from non-malignant control cells with said methylation-sensitive restriction enzyme to provide a set of control restriction fragments;    c) hybridizing under stringent conditions the test and control restriction fragments with a CpG diagnostic polynucleotide to form test and control reaction products, wherein the CpG diagnostic polynucleotide is from 35 to 3000 nucleotides in length and comprises a sequence which is identical to or substantially complementary to a target sequence that contains CpG islands that are preferentially methylated in DNA obtained from malignant cells of subjects known to have said cancer, wherein the target sequence is identified according to an RLGS process comprising:    i) digesting genomic DNA obtained from the malignant cells with the methylation-sensitive restriction enzyme of step (a) to provide a set of malignant cell restriction fragments;    ii) digesting genomic DNA obtained from non-malignant, control cells with the first restriction enzyme of step (i) to provide a set of control cell restriction fragments;    iii) attaching a detectable label to the malignant cell and control restriction fragments;    iv) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    v) separating the labeled malignant cell restriction fragments and the labeled control cell restriction fragments by a first direction electrophoresis on two different gels;    Vi) digesting the labeled malignant cell and control cell restriction fragments in each of said gels with a third restriction enzyme;    vii) electrophoresing the labeled malignant cell and control cell restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    viii) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    ix) determining the sequence of at least a portion of the diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment, and    x) determining a region of said sequence that comprise at least two CpG dinucleotides thereby determining the target sequence; and    d) assaying the size of the test and control reaction products of step c, wherein detecting a test reaction product that is larger in size as compared to a control reaction product is indicative that the cells are malignant.    
     
     
         2 . The method of  claim 1 , wherein the first restriction enzyme is NotI or AscI and the second restriction enzyme is EcoRV.  
     
     
         3 . The method of  claim 1 , wherein said target sequence is located at or near the control restriction fragment end which was cleaved by the first restriction enzyme.  
     
     
         4 . The method of  claim 1 , wherein the cancer is selected from the group consisting of: colon cancer, glioma, lung cancer, head and neck cancer, and non-medullablastoma primitive neuroectodermal tumors (PNET).  
     
     
         5 . The method of  claim 1 , wherein the diagnostic control cell restriction fragment comprises SEQ ID NO: 36.  
     
     
         6 . A method for characterizing cells isolated from a subject suspected as malignant or non-malignant, comprising: 
 a) obtaining DNA from the cells, digesting the DNA with a methylation-sensitive restriction enzyme and treating the digested DNA with a chemical compound which converts non-methylated cytosines to a different nucleotide base to obtain treated DNA;    b) reacting a portion of the treated DNA with a CpG diagnostic oligonucleotide which is complementary to a target sequence comprising CpG islands that are preferentially methylated in DNA obtained from malignant cells of subjects known to have said cancer,    c) reacting a portion of the treated DNA with a modified CpG diagnostic oligonucleotide which is complementary to a modified target sequence wherein the modified target sequence is obtained by treating the target sequence with the compound of step a to convert non-methylated cytosines to a different nucleotide base; and    d) assaying the reaction products of step b and step c to determine whether the treated    DNA has hybridized with the CpG diagnostic oligonucleotide or the modified CpG diagnostic oligonucleotide; wherein hybridization of the treated DNA with the CpG diagnostic oligonucleotide as opposed to the modified CpG diagnostic oligonucleotide indicates that cells are malignant.    
     
     
         7 . The method of  claim 6 , wherein the chemical compound is sodium bisulfite and the non-methylated cytosines are converted to uracil.  
     
     
         8 . The method of  claim 6 , wherein the target sequence is identified according to an RLGS process comprising: 
 i) digesting genomic DNA obtained from malignant cells of a subject known to have said cancer with an infrequently-cutting, methylation-sensitive, first restriction enzyme to provide a set of malignant cell restriction fragments;    ii) digesting genomic DNA obtained from non-malignant, control cells with the first restriction enzyme of step i to provide a set of control cell restriction fragments;    iii) attaching a detectable label to the malignant cell and control cell restriction fragments;    iv) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    v) separating the labeled malignant cell and control cell restriction fragments by a first direction electrophoresis on two different gels;    vi) digesting the labeled malignant cell and control cell restriction fragments in each of said gels with a third restriction enzyme;    vii) electrophoresing the labeled malignant cell and control cell restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    viii) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    ix) determining the sequence of at least a portion of the diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment, and    x) determining a region of said sequence that comprise at least two CpG dinucleotides thereby determining the target sequence.    
     
     
         9 . The method of  claim 8 , wherein the first restriction enzyme is NotI or AscI and the second restriction enzyme is EcoRV.  
     
     
         10 . The method of  claim 8 , wherein the target sequence is located at or near the control restriction fragment end that was cleaved by the first restriction enzyme.  
     
     
         11 . The method of  claim 8 , wherein the cancer is selected from the group consisting of: colon cancer, glioma, lung cancer, head and neck cancer, and non-medullablastoma primitive neuroectodermal tumors (PNET).  
     
     
         12 . The method of  claim 8 , wherein the diagnostic control cell restriction fragment comprises SEQ ID NO: 36.  
     
     
         13 . A method for characterizing cells isolated from suspected tumor tissue from a subject suspected as malignant or non-malignant, comprising: 
 a) treating DNA obtained from the cells with a chemical compound that converts non-methylated cytosines to a different nucleotide base to obtain treated DNA;    b) reacting the treated DNA with a first primer set that comprises a first and a second primer that are complementary to a first and a second sequence of a diagnostic control cell restriction fragment that has been treated by the same chemical compound as step a, wherein said first and second sequence contain non-methylated CpG islands;    c) reacting the treated DNA with a second primer set that comprises a third and a fourth primer that are complementary to a third and a fourth sequence, wherein the third and fourth sequence correspond to the same region as the first and the second sequence but contain methylated CpG islands;    wherein the sequence of a portion of the diagnostic control cell restriction fragment is obtained according to an RLGS process comprising:    i) digesting genomic DNA obtained from malignant cells of a subject known to have said cancer with an infrequently-cutting, methylation-sensitive, first restriction enzyme to provide a set of malignant cell restriction fragments;    ii) digesting genomic DNA obtained from non-malignant, control cells with the first restriction enzyme of step (i) to provide a set of control cell restriction fragments;    iii) attaching a detectable label to the malignant cell and the control cell restriction fragments;    iv) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    v) separating the labeled malignant cell and control cell restriction fragments by a first direction electrophoresis on two different gels;    vi) digesting the labeled malignant cell and control cell restriction fragments in each of said gels with a third restriction enzyme;    vii) electrophoresing the labeled malignant cell and control cell restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    viii) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    ix) determining the sequence of at least a portion of the diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment and contains CpG dinucleotides, and    d) assaying reaction products of step b and step c, wherein the existence of a reaction product with the second primer set as opposed to a reaction product with the first primer set is indicative that the treated DNA was obtained from malignant cells.    
     
     
         14 . The method of  claim 13 , wherein the chemical compound is sodium bisulfite and the non-methylated cytosines are converted to uracil.  
     
     
         15 . The method of  claim 13 , wherein the first restriction enzyme is NotI or AscI and the second restriction enzyme is EcoRV.  
     
     
         16 . The method of  claim 13 , wherein the cancer is selected from the group consisting of: colon cancer, glioma, lung cancer, head and neck cancer, and non-medullablastoma primitive neuroectodermal tumors (PNET).  
     
     
         17 . The method of  claim 13 , wherein each primer is 15-34 nucleotides in length.  
     
     
         18 . The method of  claim 13 , wherein the 3′ ends of the first and second primers comprise CpA dinucleotides.  
     
     
         19 . The method of  claim 13 , wherein the 3′ ends of the third and fourth primers comprise CpG dinucleotides.  
     
     
         20 . The method of  claim 13 , wherein the first sequence is located at or near the control restriction fragment end which was cleaved by the first restriction enzyme.  
     
     
         21 . The method of  claim 20 , wherein the first and the second sequence are from 100 to 500 base pairs apart.  
     
     
         22 . The method of  claim 21 , wherein the first and the second sequence are about 200 base pair or less apart.  
     
     
         23 . The method of  claim 13 , wherein the diagnostic control cell restriction fragment comprises SEQ ID NO 36.  
     
     
         24 . A method for characterizing suspected cells as malignant or non-malignant, comprising: 
 a) isolating suspected cells from a subject suspected of having a cancer;    b) cleaving DNA isolated from the suspected cells with a methylation-sensitive restriction enzyme, wherein the enzyme will cleave the DNA at a diagnostic recognition sequence only if the DNA is unmethylated, to provide a set of test fragments;    c) reacting the test fragments with a primer set designed to amplify a portion of a diagnostic control cell restriction fragment that contains the diagnostic recognition sequence; wherein the diagnostic recognition sequence is identified according to an RLGS process comprising:    i) digesting genomic DNA obtained from known malignant cells of said cancer comprising methylated CpG islands with the methylation-sensitive restriction enzyme of step b to provide a set of malignant cell restriction fragments;    ii) digesting genomic DNA obtained from non-malignant, control cells with the methylation-sensitive restriction enzyme of step b to provide a set of control cell restriction fragments;    iii) attaching a detectable label to the malignant cell and control cell restriction fragments;    iv) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    v) separating the labeled malignant cell and control cell restriction fragments by a first direction electrophoresis on two different gels;    vi) digesting the labeled malignant cell and control cell restriction fragments in each of said gels with a third restriction enzyme;    vii) electrophoresing the labeled malignant cell and control cell restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    viii) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    ix) determining the sequence of a portion of the diagnostic control cell restriction fragment that is located at or near an end of the fragment and comprises at least two CpG dinucleotides, thereby determining the diagnostic recognition sequence; and    d) detecting the presence of amplification products in the reaction of step c, wherein the presence of amplification products is indicative that the suspected cells are malignant.    
     
     
         25 . The method of  claim 24 , wherein the first restriction enzyme is NotI or AscI and the second restriction enzyme is EcoRV.  
     
     
         26 . The method of  claim 24 , wherein each primer in the primer set is 15-34 nucleotides in length.  
     
     
         27 . The method of  claim 24 , wherein the portion of the diagnostic control cell restriction fragment in step c is from 100 to 500 base pairs long.  
     
     
         28 . The method of  claim 27 , wherein the portion of the diagnostic control cell restriction fragment is from 100 to 200 base pairs long.  
     
     
         29 . The method of  claim 24 , wherein the cancer is selected from the group consisting of: colon cancer, glioma, lung cancer, head and neck cancer, and non-medullablastoma primitive neuroectodermal tumors (PNET).  
     
     
         30 . The method of  claim 24 , wherein the diagnostic control cell restriction fragment comprises SEQ ID NO: 36.