Promoter methylation status of hMLH1, MGMT, and CDKN2A/p16 in colorectal adenomas

Table 1 Primer sequences, annealing temperature, and product sizes for methylation-specific polymerase chain reaction assays

CpG status	Genes	Forward primer (5’→3’)	Reverse primer (5’→3’)	Genomic position2	Annealing temperature (°C)	Product size (bp)
M	hMLH1	ACGTAGACGTTTTATTAGGGTCGC	CCTCATCGTAACTACCCGCG	-716 to -602	55	115
U		TTTTGATGTAGATGTTTTATTAGGGTTGT	ACCACCTCATCATAACTACCCACA	-721 to -598	55	124
M	MGMT1	TTTCGACGTTCGTAGGTTTTCGC	GCACTCTTCCGAAAACGAAACG	+142 to +223	62	121
U		TTTGTGTTTTGATGTTTGTAGGTTTTTGT	AACTCCACACTCTTCCAAAAACAAAACA	+137 to +230	62	133
M	CDKN2A/p16	TTATTAGAGGGTGGGGCGGATCGC	CCACCTAAATCGACCTCCGACCG	+167 to +401	65	234
U		TTATTAGAGGGTGGGGTGGATTGT	CCACCTAAATCAACCTCCAACCA	+167 to +401	55	234

¹All of the primers for the MGMT gene were modified with a 20 bp GC-rich tail (5’-GCGGTCCCAAAAGGGTCAGT-3’) at their 5’ end;

²GenBank (PubMed). M: Methylated; U: Unmethylated; hMLH1: AB017806; MGMT: DD183709; CDKN2A/p16: DQ406745.

Table 2 Promoter methylation profiles for hMLH1, CDKN2A/p16, and MGMT in the four patient groups n (%)

Patient group	hMLH1 (tissue)		CDKN2A/p16 (tissue)		MGMT (tissue)		MGMT (blood)
	M	U	M	U	M	U	M	U
G I (n = 20)	6/20 (30)	14/20 (70)	3/20 (15)	17/20 (85)	9/20 (46)	11/20 (54)	8/20 (40)	12/20 (60)
G II (n = 18)	7/18 (39)	11/18 (61)	4/18 (22)	14/18 (78)	7/18 (39)	11/18 (61)	1/18 (6)	17/18 (94)
	P = 0.563		P = 0.581		P = 0.666		P = 0.0191
G III (n = 21)	10/21 (48)	11/21 (52)	14/21 (67)	7/21 (33)	16/21 (76)	5/21 (24)	7/17 (41)	10/17 (59)
	P = 0.576		P = 0.0081		P = 0.0251		P = 0.0191
G IV (n = 20)	13/20 (65)	7/20 (35)	14/20 (70)	6/20 (30)	18/20 (90)	2/20 (10)	11/18 (64)	7/18 (36)
	P = 0.279		P = 0.837		P = 0.242		P = 0.245

¹Statistically significant in comparison to the previous group. M: Methylated; U: Unmethylated.