Methylation of DAPK and THBS1 genes in esophageal gastric-type columnar metaplasia

Table 1 Sequences of specific primers used for determining gene methylation status

Primer name		Forward primer sequence (5'-3')	Reverse primer sequence (5'-3')	Product size (bp)	AT (°C)	No. of cycles
DAPK	M	GGATAGTCGGATCGAGTTAACGTC	CCCTCCCAAACGCCGA	98	60	35
	U	GGAGGATAGTTGGATTGAGTTAATGTT	CAAATCCCTCCCAAACACCAA	98	60	35
p14	M	GTGTTAAAGGGCGGCGTAGC	AAAACCCTCACTCGCGACGA	122	60	40
	U	TTTTTGGTGTTAAAGGGTGGTGTAGT	CACAAAAACCCTCACTCACAACAA	132	60	40
CDH1	M	TTAGGTTAGAGGGTTATCGCGT	TAACTAAAAATTCACCTACCGAC	115	53	35
	U	TAATTTTAGGTTAGAGGGTTATTGT	CACAACCAATCAACAACACA	97	57	35
THBS1	M	TGCGAGCGTTTTTTTAAATGC	TAAACTCGCAAACCAACTCG	74	62	40
	U	GTTTGGTTGTTGTTTATTGGTTG	CCTAAACTCACAAACCAACTCA	115	62	40

M: Methylated sequence; U: Unmethylated sequence; AT: Annealing temperature.

Table 2 Association of Helicobacter pylori cagA⁺ infection as detected by PCR, with DNA methylation of the promoter regions of target genes in the esophageal biopsies

		Helicobacter pylori infection by PCR		P value
		Negative	Positive
DAPK	Unmethylated	26	2	0.0031
	Methylated	24	16
THBS1	Unmethylated	30	5	0.0191
	Methylated	20	13
CDH1	Unmethylated	19	3	0.097
	Methylated	31	15
p14	Unmethylated	36	9	0.091
	Methylated	14	9

¹Helicobacter pylori cagA⁺ status was significantly associated with methylation.

Table 3 Bivariate analysis of the association between molecular markers and the presence of Helicobacter pylori cagA⁺ infection in esophageal biopsies

	OR	95%CI	P value
DAPK	8.67	1.80-41.7	0.0071
THBS1	3.90	1.20-12.6	0.0231
CDH1	3.06	0.78-11.9	0.110
p14	0.39	0.13-1.18	0.096

¹Statistically significant.

Table 4 Association of Helicobacter pylori cagA⁺ infection as detected by PCR with DNA methylation of the promoter regions of target genes in 33 esophageal and gastric biopsies

		Helicobacter pylori infection by PCR		P value
		Negative	Positive
Esophageal biopsies
DAPK	Unmethylated	14	0	0.0041
	Methylated	10	9
THBS1	Unmethylated	15	2	0.0571
	Methylated	9	7
CDH1	Unmethylated	11	1	0.107
	Methylated	13	8
p14	Unmethylated	18	5	0.400
	Methylated	6	4
Gastric biopsies
DAPK	Unmethylated	17	6	0.444
	Methylated	6	4
THBS1	Unmethylated	15	3	0.062
	Methylated	7	7
CDH1	Unmethylated	6	1	0.397
	Methylated	17	9
p14	Unmethylated	14	8	0.440
	Methylated	8	2

¹Statistically significant.

Table 5 Clinical and histopathological data of patients depending on the presence of Helicobacter pylori cagA⁺ infection by PCR, in esophageal biopsies

		Helicobacter pylori infection by PCR		P value
		Negative	Positive
Age (yr)	≤ 40	16	9	0.390
	41-65	14	4
	> 65	20	5
Gender	Feminine	26	7	0.340
	Masculine	24	11
Helicobacter pylori (HE)	Negative	38	8	0.0141
	Positive	12	10
Sydney Classification: Helicobacter pylori	Normal	38	8	0.0141
	Mild	11	6
	Moderate	1	2
	Marked	0	2
Sydney Classification: Neutrophils	Normal	23	5	0.0281
	Mild	25	8
	Moderate	1	4
	Marked	1	1
Sydney Classification: Mononuclear cells	Mild	17	1	0.0211
	Moderate	30	13
	Marked	3	4

¹Statistically significant. HE: Hematoxylin-eosin staining.