COLON CANCER BIOMARKER DISCOVERY

• US 20060234254A1
• Filed: 04/15/2006
• Published: 10/19/2006
• Est. Priority Date: 11/08/2004
• Status: Abandoned Application

• Current Assignee: Chiwang Yoon, Dae Kyoung Yoon, Myungsoon Kim, Sungwhan An, Tae Jeong Oh, Youngho Moon
• Original Assignee: Chiwang Yoon, Dae Kyoung Yoon, Myungsoon Kim, Sungwhan An, Tae Jeong Oh, Youngho Moon
• Inventors: Oh, Tae Jeong, Moon, Youngho, An, Sungwhan, Kim, Myungsoon, Yoon, Chiwang, Yoon, Dae Kyoung
• Application Number:
  US11/279,894
• Publication Number:
  US 20060234254A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/154 Methylation markers

Abstract

The present application discloses an epigenetic marker for colon cancer.

Claims

1. A method for discovering a methylation marker gene for the conversion of a normal cell to colon cancer cell comprising:

(i) comparing converted and unconverted cell gene expression content to identify a gene that is present in greater abundance in the unconverted cell;

(ii) treating a converted cell with a demethylating agent and comparing its gene expression content with gene expression content of an untreated converted cell to identify a gene that is present in greater abundance in the cell treated with the demethylating agent; and

(iii) identifying a gene that is common to the identified genes in steps (i) and (ii), wherein the common identified gene is the methylation marker gene.

2. The method according to claim 1, comprising reviewing the sequence of the identified gene and discarding the gene for which the promoter sequence does not have a CpG island.

3. The method according to claim 1, wherein the comparing is carried out by direct comparison.

4. The method according to claim 1, wherein the comparing is carried out by indirect comparison.

5. The method according to claim 1, wherein the demethylating agent is 5 aza 2′

-deoxycytidine (DAC).

6. The method according to claim 1, comprising confirming the methylation marker gene, which comprises assaying for methylation of the common identified gene in the converted cell, wherein the presence of methylation in the promoter region of the common identified gene confirms that the identified gene is the marker gene.

7. The method according to claim 6, wherein the assay for methylation of the identified gene is carried out by

i. identifying primers that span a methylation site within the nucleic acid region to be amplified,

ii. treating the genome of the converted cell with a methylation specific restriction endonuclease,

iii. amplifying the nucleic acid by contacting the genomic nucleic acid with the primers, wherein successful amplification indicates that the identified gene is methylated, and unsuccessful amplification indicates that the identified gene is not methylated.

8. The method according to claim 7, wherein the converted cell genome is treated with an isoschizomer of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG-sites as a control.

9. The method according to claim 7, wherein detecting the presence of amplified nucleic acid is carried out by hybridization with a probe.

10. The method according to claim 9, wherein the probe is immobilized on a solid substrate.

11. The method according to claim 7, wherein the amplification is carried out by PCR, real time PCR, or amplification or linear amplification using isothermal enzyme.

12. The method according to claim 1, wherein detection of methylation on the outer part of the promoter is indicative of early detection of cell conversion.

13. A method of identifying a converted colon cancer cell comprising assaying for the methylation of the marker gene identified in claim 1.

14. A method of diagnosing colon cancer or a stage in the progression of the cancer in a subject comprising assaying for the methylation of the marker gene identified using the method in claim 1.

15. The method according to claim 14, wherein the marker gene is LAMA2 (NT_—

025741)—

laminin alpha2(merosin, congenital);

FABP4 (NT_—

008183)—

Adipocyte acid binding protein 4;

GSTA2 (NT_—

007592)—

glutathione S transferase A2;

STMN2 (NT_—

008183)—

Stathmin-like 2;

NR4A2 (NT_—

005403)—

Nuclear receptor subfamily 4 group A, member 2;

DSCR1L1 (NT_—

007592)—

Down syndrome cadidate region gene 1 like-1;

AMBP (NT_—

008470)—

alpha-1-microglobulin/bikunin precursor;

SEPP1 (NT_—

006576)—

selenoprotein P, plasma 1;

ID3 (NT_—

004610)—

inhibitor of DNA binding 3, dominant negative helix-loop-helix protein;

RGS2 (NT_—

004487)—

regulator of G-protein signalling 2;

WISP2 (NT_—

011362)—

WNT1 inducible signaling pathway protein 2;

MGLL (NT_—

005612)—

monoglyceride lipase;

CPM (NT_—

029419)—

carboxypeptidase M 12q14.3;

GABRA1 (NT_—

023133)—

gamma-aminobutyric acid (GABA) A receptor, alpha 1;

CLU (NT_—

023666)—

clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J); and

F2RL1 (NT_—

006713)—

coagulation factor II (thrombin) receptor-like 1, or a combination thereof.

16. A method of diagnosing likelihood of developing colon cancer comprising assaying for methylation of a colon cancer specific marker gene in normal appearing bodily sample.

17. The method of claim 16, wherein the marker gene is LAMA2 (NT_—

025741)—

laminin alpha2(merosin, congenital);

FABP4 (NT_—

008183)—

Adipocyte acid binding protein 4;

GSTA2 (NT_—

007592)—

glutathione S transferase A2;

STMN2 (NT_—

008183)—

Stathmin-like 2;

NR4A2 (NT_—

005403)—

Nuclear receptor subfamily 4 group A, member 2;

DSCR1L1 (NT_—

007592)—

Down syndrome cadidate region gene 1 like-1;

AMBP (NT_—

008470)—

alpha-1-microglobulin/bikunin precursor;

SEPP1 (NT_—

006576)—

selenoprotein P, plasma 1;

ID3 (NT_—

004610)—

inhibitor of DNA binding 3, dominant negative helix-loop-helix protein;

RGS2 (NT_—

004487)—

regulator of G-protein signalling 2;

WISP2 (NT_—

011362)—

WNT1 inducible signaling pathway protein 2;

MGLL (NT_—

005612)—

monoglyceride lipase;

CPM (NT_—

029419)—

carboxypeptidase M 12q14.3;

GABRA1 (NT_—

023133)—

gamma-aminobutyric acid (GABA) A receptor, alpha 1;

CLU (NT_—

023666)—

clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J); and

F2RL1 (NT_—

006713)—

coagulation factor II (thrombin) receptor-like 1, or a combination thereof.

18. The method according to claim 16, wherein the bodily sample is solid tissue, stool, or body fluids.

19. The method according to claim 16, wherein likelihood of developing colon cancer is determined by reviewing a panel of colon-cancer specific methylated genes for their level of methylation and assigning level of likelihood of developing colon cancer.