Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis

Table 1 Clinical-pathologic profiles of the CRC cases in this study

Clinical-pathologic features		Number of cases
Gender	Male	37
	Female	28
Age (yr)	Median	60
	Range	28-85
Tumor site	Left colon and rectum	43
	Right colon	22
Histological	Adenocarcinoma	51
types	Mucinousi carcinoma	5
	Other types combined1	9
Differentiation Moderate	51
	Poor	14
Follow-up	Survival	27
(3 yr)	Deceased	9
Duke’s stage	A	6
	B	15
	C	30
	D	14

¹Other types combined included the mixed squamous cell carcinoma, carcinoid carcinoma, etc.

Table 2 Clinical-pathologic profiles of adenomas and neigh-boring tumor-free mucosae

Clinical-pathologic features		Adenoma	Mucosa
Gender	Male	4	2
	Female	4	4
Age (yr)	Median	60	68
	Range	34-83	47-76
Tumor site	Left colon	6	4
	Right colon	2	2
Histological	Tubular	3
type	Tubulovillous	5
Dysplasia	Light-moderate	8

Table 3 Target CpG islands and the primers for MSP

Gene	GenBank No.	Primer		Location to transcription start	Size (bp)
		Sense (5’-3’)	Anti-sense (5’-3’)
APC	U02509	(U) GTGTTTTATTGTGGAGTGTGGGTT	CCAATCAACAAACTCCCAACAA	-17 147 to -17 050	108
		(M) TATTGCGGAGTGCGGGTC	TCGACGAACTCCCGACGA	-17 153 to -17 046	97
BRCA1	L78833	(U) GGTTAATTTAGAGTTTTGAGAGATG	TCAACAAACTCACACCACACAATCA	-320 to -138	182
		(M) GGTTAATTTAGAGTTTCGAGAGACG	TCAACGAACTCACGCCGCGCAATCG	-320 to -138	182
CDH1	NM_004938	(U) GGTGGGTGGGTTGTTAGTTTTGT	AACTCACAAATCTTTACAATTCCAAC	-266 to -93	172
		(M) GTGGGCGGGTCGTTAGTTTC	CTCACAAATACTTTACAATTCCGACG	-265 to -93	172
CDH13	AB001090	(U) TTGTGGGGTTTGTTTTTTGT	AACTTTTCATTCATACACACA	-267 to -24	243
		(M) TCGCGGGGTTCGTTTTTCGC	GACGTTTTCATTCATACACGCG	-267 to -24	243
CDX1	NT_029289	(U) TGATTGGGTTGTTGTTTATGG	TACCACCACCACCTCCAA	+290 to +522	233
		(M) CGATTGGGTCGTCGTTTA	CCGCATCCACTCGTAAAA	+290 to +491	202
COX2	NT_004487	(U) TTGTTTGTTGTTGTGATGTTTG	TCCAAACTCTTTCCCAAATC	-119 to -324	206
		(M) GTTCGTCGTTGCGATGTT	CCAAACTCTTTCCCAAATCA	-121 to -323	203
CXX1	NT_011786	(U) TTAGGTTGGTTTTTGTGGATATG	CACCCAACCATCCATCAC	-24 to +92	117
		(M) AGGTCGGTTTTCGTGGAT	GATCGATATCGCCGTCAA	-22 to +192	215
Cyclina1	AF124143	(U) GGGTAGTTTTGTTGTGTTTTAGTTG	AACCACTAACAACCCCCTCT	-762 to -565	199
		(M) TCGTCGCGTTTTAGTCGT	ACCCGTTCTCCCAACAAC	-755 to -550	206
DAPK1	NM_004938	(U) GGATAGTTGGATTGAGTTAATGTC	CAAATCCCTCCCAAACACCAA	-332 to -229	103
		(M) GGATAGTCGGATCGAGTTAACGTC	CCCTCCCAAACGCCGA	-332 to -234	98
DNMT1	NT_011176	(U) GGGTGGTAGATGTTGTTTTTGT	CACCCTACCTATCCCCCTAA	-359 to -130	230
		(M) CGTCGTTTTCGTTTATCGTT	ATACCTACCGCCTACGAACA	-320 to -140	181
hMLH1	AB017806	(U) TTTTGATGTAGATGTTTTATTAGGGTTGT	ACCACCTCATCATAACTACCCACA	-526 to -654	118
		(M) ACGTAGACGTTTTATTAGGGTCGC	CCTCATCGTAACTACCCGCG	-531 to -655	124
MAGEA1	U82670	(U) GTTTGGTTGAAGGAATTTGA	ACCCACAACCCTCCCTCTTA	+24 to +347	324
		(M) GTTCGGTCGAAGGAATTTGA	CCACAACCCTCCCTCTTAAA	+24 to +345	322
MGMT	AL355531	(U) TTTGTGTTTTGATGTTTGTAGGTTTTTGT	AACTCCACACTCTTCCAAAAACAAAACA	-451 to -266	209
		(M) TTTCGACGTTCGTAGGTTTTCGC	GCACTCTTCCGAAAACGAAACG	-469 to –261	186
MINT1	AF135501	(U) TATTTTTGAAGTGTTTGTTTGGTGT	TCCCTCTCCCCTCTAAACTTC		202
		(M) TTCGAAGCGTTTGTTTGG	CGCCTAACCTAACGCACA	*	160
MINT31	AF135531	(U) GGGTGGGAATTGAGATGATT	CATCACCACCCCTCACTTTA		131
		(M) GCGGGAATTGAGACGATT	ACGCTTACGCCACTACGA	*	176
MYOD1	NT_009307	(U) ATTTGATGGTTTTTGATGGTTT	CACACACATACTCATCCTCACA	+206 to +418	213
		(M) GACGGTTTTCGACGGTTT	GCCCGAAACCGAATACAC	+210 to +393	184
N33	NT_030737	(U) TTTGGTGAATTGGATGTTTTG	CACCCAACTCCTACCACACAC	+20 to +169	150
		(M) TTTTCGGTGAATCGGATG	TACGCGCCCAACTCCTA	+18 to +173	156
p14ARF	L41934	(U) TGAGTTTGGTTTTGGAGGTGG	AACCACAACAACAAACACCCCT	+97 to +262	165
		(M) GTCGAGTTCGGTTTTGGAGG	AAAACCACAACGACGAACG	+95 to +255	160
p15INK4b	NM_004936	(U) TGTGATGTGTTTGTATTTTGTGGTT	CCATACAATAACCAAACAACCAA	-318 to -164	154
		(M) GCGTTCGTATTTTGCGGTT	CGTACAATAACCGAACGACCGA	-312 to -165	147
p16INK4a	NM_000077	(U) TTATTAGAGGGTGGGGTGGATTGT	CAACCCCAAACCACAACCATAA	-80 to +71	151
		(M) TTATTAGAGGGTGGGGCGGATCGC	ACCCCGAACCGCGACCGTAA	-80 to +69	149
p21WAF1	NT_007592	(U) TTTTTGTAGTATGTGAGGTTTTGG	AACACAACTCAACACAACCCTA	-200 to -1	200
		(M) TGTAGTACGCGAGGTTTCG	TCAACTAACGCAACTCAACG	-196 to +5	202
p27KIP1	AB003688	(U) TGTGATTTTGATGTTGGTAAGGT	CAAACCACAACCCAAACTCT	-363 to -141	223
		(M) CGACGTCGGTAAGGTTTG	AAACGCGCAAAAACTACG	-355 to -163	193
p73	AB031234	(U) TGGGTGTTTGGTTTGTAGGT	CCAACTCTCAACTCCCAAAA	-1 725 to -1 505	221
		(M) GCGTTCGGTTCGTAGGTT	CTCAACTCCCAAAACCCAA	-1 722 to -1 511	212
PTEN	NM_000314	(U) TGGTTTTTTGAGGTGTTTG	TTCCATCATAACTACAACTTCCA	-979 to -812	167
		(M) GGTTTTTCGAGGCGTTCG	CGCCTCACAACGACTCAACT	-978 to -786	192
RAR-β	NM_016152	(U) TTGAGAATGTGAGTGATTTGA	AACCAATCCAACCAAAACAA	-343 to -197	146
		(M) TCGAGAACGCGAGCGATTCG	GACCAATCCAACCGAAACGA	-343 to -197	146
RASSF1A	NT_022517	(U) TTTGGTTGGAGTGTGTTAATGTG	CAAACCCCACAAACTAAAAACAA	+70 to +178	108
		(M) GTGTTAACGCGTTGCGTATC	AACCCCGCGAACTAAAAACGA	+82 to +176	94
RASSF1C	NT_022517	(U) GGAGTTTGGATTGTTGGTTTTG	CACCCCCAAAAATAACCTCAT	-370 to -137	187
		(M) AGTTTGGATTGTCGGTTTCG	TCACAAACCCCACCTACCAC	-370 to -137	187
SFRP1	NT_008251	(U) GTTTTGTGGTTGTAAGTTGTTGTT	AAACCCCACACACTCCAA	-180 to +19	199
		(M) TCGCGGTCGTAAGTTGTT	CGCACTCCAACCCTACAA	-177 to +11	188
TIMP3	NM_000362	(U) TTTTGTTTTGTTATTTTTTGTTTTTGGTTTT	CCCCCAAAAACCCCACCTCA	-454 to -335	119
		(M) CGTTTCGTTATTTTTTGTTTTCGGTTTC	CCGAAAACCCCGCCTCG	-451 to -335	116
VHL	AF010238	(U) GTTGGAGGATTTTTTTGTGTATGT	CCCAAACCAAACACCACAAA	-185 to -20	165
		(M) TGGAGGATTTTTTTGCGTACGC	GAACCGAACGCCGCGAA	-183 to -25	158
WT1	X74840	(U) TGGGATTTGGGTGGTATTTG	CACCAACACCCACTACACCA	+295 to +510	216
		(M) GTTAGGCGTCGTCGAGGTTA	AAAACGCAAAATCCAACACC	+321 to +526	206

Table 4 Antibodies for immunohistochemical analysis

Antibody name	Expression location	Company	Catalogue No.
CDH1	Membrane/Cytoplasm	Antibody Diagnostica INC.	M-0536
CDH13	Cytoplasm	Santa Cruz	sc-7940
COX2	Cytoplasm	Santa Cruz	sc-1746
Cyclin a1	Nuclear	Novocastra Lab.	NCL-CYCLINA
hMLH1	Nuclear	PharMingen	G168-15
MGMT	Cytoplasm	DAKO	M3610
p14ARF	Nulear/Cytoplasm	Santa Cruz	sc-8340
p73	Cytoplasm	Santa Cruz	sc-17823
RAR	Nuclear/ Cytoplasm	Santa Cruz	sc-552
TIMP3	Cytoplasm	Oncogene	IM43T

Table 5 Expression profile of the genes in the context of DNA methylation status

Methylation status	Positive rate % (Positive cases/the total cases)
	CDH13	Cyclin a1	RAR-	COX2	P73	MGMT	hMLH1	p14ARF	TIMP3	CDH1
U	73 (16/22)	/	100 (8/8)	81 (35/43)	60 (12/20)	24 (11/46)	74 (35/47)	100 (51/51)	80 (46/58)	98 (57/58)
U/M	50 (14/28)	70 (37/53)	79 (22/28)	93 (14/15)	54 (19/35)	25 (2/8)	36 (4/11)	100 (2/2)	/	/
M	38 (3/8)	80 (4/5)	85 (17/20)	/	/	50 (2/4)	/	/	/	/
Total	57 (33/58)	71 (41/58)	84 (47/56)	84 (49/58)	53 (31/58)	26 (15/58)	67 (39/58)	100 (53/53)	79 (46/58)	98 (57/58)
Non-cancerous mucosa	+	/	++	/	+	+	++	+	+	++