Method and nucleic acids for the analysis of a colon cell proliferative disorder

Abstract

The present invention relates to modified and genomic sequences, to oligonucleotides and/or PNA-oligomers for detecting the cytosine methylation state of genomic DNA, as well as to a method for ascertaining genetic and/or epigenetic parameters of genes for use in the differentiation, diagnosis, treatment and/or monitoring of colon cell proliferative disorders, or the predisposition to colon cell proliferative disorders.

1 . A gene panel, comprising at least one target nucleic acid from the group of genes and/or their regulatory regions comprising MDR1, APOC2, CACNA1G, EGR4, AR, RB1, GPIb beta, MYOD1, WT1, HLA-F, ELK1, APC, BCL2, CALCA, CDH1, CDKN1A, CDKN1B (p27 Kip1), CDKN2a, CDKN2B, CD44, CSPG2, DAPK1, EGFR, EYA4, GSTP1, GTBP/MSH6, HIC-1, HRAS, IGF2, LKB1, MGMT, MLH1, MNCA9, MSH3, MYC, N33, PAX6, PGR, PTEN, RARB, SFN, S100A2, TGFBR2, TIMP3, TP53, TP73, VHL, CDKN1C, CAV1, CDH13, DRG1, PTGS2, THBS1, TPEF (=TMEFF2; =HPP1), DNMT1, CEA, MB, PCNA, CDC2, ESR1, CASP8, RASSF1, MSH4, MSH5, and wherein at least one target nucleic acid is selected from the gene CD44 or GPIb beta and/or their regulatory regions.  
     
     
         2 . The gene panel according to  claim 1 , wherein the target nucleic acids are selected from the group of genes and/or their regulatory regions comprising APC, CALCA, CAV1, CD44, CDH1, CDH13, CDKN2a, CSPG2, DAPK1, EGFR, EGR4, ESR1, GSTP1, GTBP/MSH6, HLA-F, IGF2, LKB1, MLH1, MYOD1, N33, PTEN, PTGS2, TGFBR2, TP73, TPEF (=TMEFF2; —HPP1), WT1, and EYA4, and wherein at least one target nucleic acid is selected from the gene CD44 and/or its regulatory regions.  
     
     
         3 . The gene panel according to  claim 1 , wherein the target nucleic acids are selected from the group of genes and/or their regulatory regions comprising APC, AR, BCL2, CALCA, CAV1, CD44, CDH1, CDH13, CDKN2a, CEA, CSPG2, DAPK1, EGFR, EGR4, ESR1, GPIb beta, GSTP1, GTBP/MSH6, HIC-1, HLA-F, IGF2, LKB1, MGMT, MLH1, MSH3, MYC, MYOD1, N33, PCNA, PGR, PTEN, PTGS2, RARB, RASSF1, S100A2, TGFBR2, TP73, TPEF (=TMEFF2; =HPP1), WT1, and EYA4, and wherein at least one target nucleic acid is selected from the gene CD44 and/or its regulatory regions.  
     
     
         4 . The gene panel according to  claim 1 , wherein the target nucleic acids are selected from the group of genes and/or their regulatory regions comprising APC, CAV1, CD44, CDH13, CSPG2, EGFR, GSTP1, HLA-F, IGF2, N33, PTEN, PTGS2, TP73, TPEF (=TMEFF2; =HPP1), and EYA4, and wherein at least one target nucleic acid is selected from the gene CD44 and/or its regulatory regions.  
     
     
         5 . The gene panel according to  claim 1 , wherein said target nucleic acids are selected from the group of genes and/or their regulatory regions consisting of GPIb beta and CDKN2a.  
     
     
         6 . The gene panel according to any of  claims 1  to  5 , wherein said target nucleic acids are selected from the group comprising SEQ ID NO: 133 to SEQ ID NO: 388 and sequences complementary thereto.  
     
     
         7 . The gene panel according to any of  claims 1  to  6 , wherein said panel is present in the form of a nucleic acid or peptide nucleic acid array for the analysis of colon cell proliferative disorders associated with the methylation state of said target nucleic acids.  
     
     
         8 . The array according to  claim 7 , characterised in that a solid phase surface of said array is composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold.  
     
     
         9 . An oligonucleotide array, characterised in that said array comprises at least one oligomer, in particular an oligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomer comprising at least one base sequence of at least 10 nucleotides which hybridises to or is identical to a pretreated genomic DNA according to one of the SEQ ID NO: 133 to SEQ ID NO: 388, and wherein at least one target nucleic acid is selected from the pretreated genomic DNA of the gene CD44 or GPIb beta and/or their regulatory regions.  
     
     
         10 . The oligonucleotide array according to  claim 9 , wherein the base sequence of the oligonucleotides includes at least one CpG or TpG dinucleotide sequence.  
     
     
         11 . The oligonucleotide array according to  claim 10 , wherein the cytosine of said at least one CpG or TpG dinucleotide is/are located in the middle third of the oligomer.  
     
     
         12 . The oligonucleotide array according to any of  claims 9  to  11 , wherein the base sequence of the oligonucleotides is selected from the group of SEQ ID NO: 519 to SEQ ID NO: 1030.  
     
     
         13 . The oligonucleotide array according to any of  claims 9  to  12 , comprising a set of oligonucleotides selected from the group of at least two oligonucleotides according to SEQ ID NO: 986 to 895, at least two oligonucleotides according to SEQ ID NO: 895 to 906, 909 to 918, 921 to 924, 931, 932, 941, 942, 971, 972, and 987 to 990, at least two oligonucleotides according to SEQ ID NO: 895 to 954, 957 to 962, 965 to 970, 975 to 978, 981 to 986, and 991 to 1028, and at least two oligonucleotides according to SEQ ID NO: 1005, 1006, 1029, and 1030.  
     
     
         14 . A method for detecting and differentiating between colon cell proliferative disorders, comprising the following steps; 
 a) providing a biological sample containing genomic DNA,    b) extracting said genomic DNA,    c) converting cytosine bases in said genomic DNA sample which are unmethylated at the 5-position, by treatment, to uracil or another base which is dissimilar to cytosine in terms of base pairing behaviour,    d) providing a panel or an array according to any of  claims 1  to  13 , and    e) identifying the methylation status of one or more cytosine positions based on said array.    
     
     
         15 . The method according to  claim 14 , characterised in that the reagent is a solution of bisulfite, hydrogen sulfite or disulfite.  
     
     
         16 . The method according to  claim 14  or  15 , characterised in that the fragments of said pretreated genomic DNA are amplified by means of the polymerase chain reaction CPCR) prior to step (d).  
     
     
         17 . The method according to any of  claims 14  to  16 , characterised in that more than ten different fragments having a length of 100-2000 base pairs are amplified.  
     
     
         18 . The method according to any of  claims 14  to  17 , characterised in that the amplification step is carried out using a set of primer oligonucleotides comprising SEQ ID NO: 389 to SEQ ID NO: 518.  
     
     
         19 . The method according to any of  claims 14  to  18 , characterised in that the amplification step preferentially amplifies DNA which is of particular interest in healthy and/or diseased colon tissues, based on the specific genomic methylation status of colon tissue, as opposed to background DNA.  
     
     
         20 . The method according to any of  claims 14  to  19 , characterised in that identifying the methylation status of one or more cytosine positions based on said array involves a hybridisation of each amplificate to an oligonucleotide or peptide nucleic acid (PNA)-oligomer.  
     
     
         21 . The method according to  claim 20 , characterised in that the amplificates are labelled.  
     
     
         22 . The method according to  claim 21 , further comprising detecting the amplificates or fragments of the amplificates by mass spectrometry.  
     
     
         23 . A method for detecting and differentiating between colon cell proliferative disorders, comprising the following steps; 
 a) providing a biological sample containing genomic DNA,    b) extracting said genomic DNA,    c) providing a gene panel according to any of  claims 1  to  6 ,    d) digesting the genomic DNA according to said gene panel with one or more methylation sensitive restriction enzymes, and    e) detection of the DNA fragments generated in the digest of step d).    
     
     
         24 . The method according to  claim 23 , wherein the DNA digest is amplified prior to Step e).  
     
     
         25 . The method according to  claim 24 , wherein the amplification is carried out by means of the polymerase chain reaction (PCR).  
     
     
         26 . The method according to  claim 24  or  25 , wherein the amplification of more than one DNA fragments is carried out in one reaction vessel.  
     
     
         27 . The method according to any of  claims 14  to  26 , wherein said method differentiates between normal colon tissue and colon cell proliferative disorder tissue, differentiates between colon adenoma tissue and normal colon tissue, differentiates between colon carcinoma tissue and normal colon tissue or differentiates between colon adenoma tissue and colon carcinoma tissue.  
     
     
         28 . A kit comprising a bisulfite (=disulfite, hydrogen sulfite) reagent as well as oligonucleotides and/or PNA-oligomers comprising at least one base sequence of at least 10 nucleotides which hybridises to or is identical to a pretreated genomic DNA according to one of the SEQ ID NO: 133 to SEQ ID NO: 388, and wherein at least one base sequence is selected from the pretreated genomic DNA of the gene CD44 or GPIb beta and/or their regulatory regions.  
     
     
         29 . Use of a gene panel according to any of  claims 1  to  6  or of an array according to any of  claims 7  to  13  for the detection of a predisposition to, differentiation between subclasses, diagnosis, prognosis, treatment, and/or monitoring of colon cell proliferative disorders.