Methylation Status of CDH1 Gene in Gastric Biopsy Specimens and Its Association with H. Pylori Infection

Abstract

Background:

Helicobacter pylori (H. pylori) have been accepted as having an etiologic role in gastro-duodenal diseases as chronic gastritis, peptic ulcer, and gastric carcinoma. Methylation of CGI has been correlated with the tumorigenic process since it can inactivate tumor suppressor genes. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is preserving cell-cell connections. Early stages of gastric carcinogenesis may be affected by the promoter methylation-mediated inactivation of this gene.

Objective:

This study aimed to investigate the methylation status of CDH1 using Methylation-Specific PCR (MSP) technique in clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy and correlated it with H. pylori detection by glmM PCR test.

Methods:

Fifty gastric mucosal biopsies were selected from one hundred and five samples included in this study. The detection of H. pylori was performed with the PCR primers specific to glmM gene. Bisulfite modification was done and the methylation status of the CDH1 gene was detected using MSP reaction.

Results:

H. pylori was detected in 36% (18/50) of study population using glmM gene PCR test, 89% (16/18) of H. pylori positive cases were CDH1 methylated positive (chi-square, p-value=0.002). CDH1 methylation can be present in cancerous and noncancerous gastric mucosa, where 60% (18/30) of CDH1 methylation positive gastric mucosa showed gastritis as an endoscopy finding and gastric cancer in 6% (2/30). There was a significant correlation between and CDH1 methylation positive results and age group (P-value = 0.02). There was no significant correlation between CDH1 methylation positive results and participants gender (p-value=0.431) and clinical symptoms (all P-value > 0.05).

Conclusion:

This work suggested strong significance association between H. pylori infection and CDH1 methylation.

Introduction

In mammals, DNA methylation occurs when a methyl or hydroxymethyl group is added to the cytosine at position C5, forming 5-methylcytosine., It mostly only occurs in CpG dinucleotide (Kurdyukov and Bullock, 2016; Schubeler, 2015). DNA methylation is a major epigenetic mechanism and plays a critical part in numerous important biological processes such as embryonic development, gene imprinting, and gene expression regulation. Aberrant DNA methylation has been demonstrated to be a mechanism for the development of many malignancies and is linked to further complicated disorders. (e.g diabetes and cardiovascular diseases, neurodegenerative and psychiatric disorders) (Jin and Liu, 2018; Nakajima et al., 2006).

Methylation of CpG islands has been attributed to the tumorigenic process because it can inactivate tumor suppressor genes (Zhang et al., 2014). Therefore, DNA methylation profiles are considered as markers for disease assessment, prognosis, and response to treatment (Šestáková et al., 2019).

Gastric cancer (GC) is one of the most prevalent cancers in the world; it continues to be the major cause of cancer death in Asia and certain European countries. As the main cause of death among malignant tumors, gastric carcinoma has an average yearly mortality rate of up to 16 per 100,000 people (Jemal et al., 2004). Gastric cancer is known to be associated with tumor-suppressor or tumor-related genes that are inactivated more frequently by CGI hyper methylation than by mutations. Additionally, CGI hyper methylation has been observed in the non-neoplastic gastric mucosa of persons without GC as well as in the surrounding non-cancerous tissues of GC patients (Shin et al., 2010). Helicobacter pylori colonize the human gastrointestinal tract and has been acknowledged as contributing to the development of gastro duodenal disorders such chronic active gastritis, peptic ulcers, and gastric cancer. It has been classified by the International Agency for Research on Cancer (IARC) as a grade-I carcinogen (Ohata et al., 2004; Ahsan et al., 2008; Fakhrjou et al., 2011). Numerous publications have revealed a connection between certain gene promoter methylation and H. pylori infection (Perri et al., 2007; Kaise et al., 2008). Promoter CGI hypermethylation appears to be triggered by active inflammation due to H. pylori (Ushijima, 2007; Chan et al., 2006; Qian et al., 2008). It is not yet known, nevertheless, whether persistent aberrant DNA hypermethylation will remain after the active H. pylori infection stops (Shin et al., 2008). CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is a critical in maintaining cell-cell contacts. The chance of metastasis may rise if the CDH1 gene is inactivated. A calcium-dependent transmembrane adhesion glycoprotein called E-cadherin is codified by CDH1 (Tamura, 2002). Epithelial cells produce the tumor suppressor gene CDH1, which is crucial for maintaining tissue integrity and cellular activities such as adhesion, morphology, migration, and development (Ashour et al., 2002). The inactivation of CDH1 gene is related with cancer growth and involving in invasion and metastasis and it is seen in a variety of cancers, including gastric cancer (Bartchewsky et al., 2009). Gastric cancer of the diffuse histological type carries a CDH1 mutation in about 50% of cases. Moreover, CDH1 hyper methylation was discovered to be the second factor in two kin with familial gastric cancer and a germ line CDH1 mutation (Kague et al., 2010). This study aimed to investigate the methylation status of CDH1 using MSP technique in clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy and correlated it with H. pylori detection by glmM gene PCR method.

Materials and Methods

Patients and specimens

Fifty gastric mucosal biopsies from 105 clinically suspected patients for H. pylori infection were included in this study. The samples selected according to concentration of DNA, which should be more than 200 µg. The specimens were collected from endoscopy unit, Wad Medani Teaching Hospital, Gezira state, Sudan, between March 2018 and December 2019. The presence of H. Pylori was detected by: histopathology and (glmM) gene PCR analysis in gastric biopsies. In histopathology we used hematoxylin and eosin stain and Warthin-Starry stains. We calculated the sensitivity, specificity, negative predictive value and positive predictive value of each method for detection of H. Pylori. We depended on glmM gene molecular method as a gold standard for detection of H. pylori (the sensitivity was 92%). All molecular analyses were performed in the Molecular Biology Laboratory, Faculty of Medical Laboratory Sciences, University of Gezira.

Molecular identification

DNA extraction and PCR of glmM gene

The innuPREP DNA Mini Kit DNA extraction protocol (analyticjena, Germany, LOT:023-17, REF:845-KS-1040050) was followed for extraction of DNA from gastric biopsy. Extracted genomic DNA with concentration more than 200 µg was amplified using glmM H pylori specific primers. Primer sequences, amplicon sizes and the annealing temperature are showed in Table 1. PCR reactions were performed in a volume of 20 µL containing 5 µL genomic DNA, 1.5 µL forward primer (10µM in concentration), 1.5 µL reverse primer (10µM in concentration), 5 µL master mix and 7µl Deionized sterile water. Cycling for primers was as follows: primary denaturation (94°C for 5 min) then 30 cycles of alternating denaturation (94°C for 45 sec), annealing of primers (60°C for 45 sec) and extension by the thermo-stable polymerase (72°C for 1 min) with final extension of 10 min. The resulted amplicon was visualized by UV in a 2% agarose gel electrophoresis method.

Table 1.

Primer Sequences, Amplicon Sizes and the Annealing Temperature Used in This Study

Primer	Primer sequence (5´-3´) forward Primer sequence (5´-3´) reverse	Product size (bp)	Annealing T (°C)	Reference
glmM	5’- AAGCTTTTAGGGGTGTTAGGGGTTT-3´	294	60	(Bickley et al., 1993)
	5´-AAGCTTACTTTCTAACACTAACGC-3´
E-cadherin methylated	5’-TTA-GGT-TAG-AGG-GTT-ATC-GCG-T-3´	115	62	(Kague et al., 2010)
	5’-TAA-CTA-AAA-ATT-CAC-CTA-CCG-AC– 3’
E-cadherin unmethylated	5’- TAA-TTT-TAG-GTT-AGA-GGG-TTA-TTG-T– 3’ 5’- CAC-AAC-CAA-TCA-ACA-ACA-CA– 3’	97	58

CDH1 methylation technique

Bisulfite modification

Purified DNA was subjected to bisulfite modification. Bisulfite treatment converts unmethylated cytosine in uracil and methylated cytosine is unchanged. Briefly, 20 μl of DNA sample was added to 130 μl of the CT Conversion Reagent in a PCR tube then Placed in a thermal cycler at 98°C for 10 minutes followed by 64°C for 2.5 hours. 600 μl of M-Binding Buffer was added to a Zymo-Spin™ IC Column and place the column into a provided Collection Tube then Loaded the sample into the Zymo-Spin™ IC Column containing the M-Binding Buffer. Centrifuge at full speed (>10,000 x g) for 30 seconds. Discard the flow-through. 100 μl of M-Wash Buffer was added to the column. Centrifuge at full speed for 30 seconds. 200 μl of M-Desulphonation Buffer was added to the column and let stand at room temperature (20-30°C) for 15-20 minutes, centrifuge at full speed for 30 seconds. 400 μl of M-Wash Buffer was added to the column. Centrifugation and elution the DNA by adding 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed. The DNA was stored below -20°C. (ZYMO RESEARCH CORP. EZ DNA Methylation-Gold™ Kit Catalog Nos. D5005 & D5006).

MSP (methylation-specific PCR) for CDH1 gene

Bisulfite-modified DNA was amplified in two PCR reactions. The first reaction with primers specific for methylated gene, and the second one for unmethylated gene. All primer sequences, product size and annealing temperatures were summarized in Table 1. PCR was performed in 20μL reaction volumes, containing 1µl of Bisulfite-modified DNA, master mix 4µl, foreword primer 0.5µl, reveres primer 0.5µl and the reaction volume was completed to 20µl by adding 14µ Deionized sterile water. Amplicons were screened using 2% agarose gel and gel documentation system.

Statistical analysis

Statistical analysis was performed using SPSS (IBM Corp. Released 2017. IBM SPSS Statistics for windows, Version18.0 Armonk, NY: IBM Corp)

The descriptive statistics of categorical variables were presented as frequency and percentage. Chi-squared tests were used for categorical variables. Associations between variables were examined by using Pearson correlation.

Results

Patient characteristics

This study included fifty participants from a total of one hundred and five clinically suspected patients for H. pylori infection who undergoing upper gastrointestinal endoscopy. Among these, 46% were women and 54% were men. The age range between 20- 72 years with the mean age 47 years and SD 16.

Most study population suffering from Epigastric pain (76%) followed by vomiting in 44% of patients followed by lack of appetite in 38%, gastric reflex and nausea in 32%, other symptoms were founded among participants includes (black feces, chest pain, vomiting of blood and dysphagia). Endoscopy findings diagnosed 62% of patients as chronic gastritis, 22% had esophagitis, 6%, had stomach cancer, 4% had duodenal ulcer and normal endoscopy result in 6%.

Detction of H. pylori in the study population using glmM gene

Detection of H. pylori was found in 18 (36%) of study population, 94% of them show gastritis as an endoscopy finding and 6% showed gastric cancer.

Methylation status of CDH1 Gene

Using MSP technique the methylation of CDH1 using methylated primers was observed in 30 (60%) of study samples while 20 (40%) of study samples showed negative results. Unmethylated primers gave positive results in 10 (20%) of study samples (Figure 1). Applying cross tabulation analysis: 27 samples gave positive results with methylated primer and negative to unmethylated primer, 7 samples positive with unmethylated and negative with methylated primer. 3 samples gave positive results with both primers, 13 samples negative with the both primers (Table 2).

Figure 1.

Gel Electrophoresis Representative MSP PCR Products of CDH1Gene. DNA ladder = 100 bp, samples 1 was positive in both methylated (M) and unmethylated (U), sample 2, 3 and 4 were positive just only in methylated (M)

Table 2.

Methylated * Unmethylated Cross Tabulation

		Unmethylated		Total
		Positive	Negative
Methylated	Positive	3	27	30
	Negative	7	13	20
Total		10	40	50

CDH1 methylation and H. pylori

Among H. pylori positive cases 89% (16 from 18) were CDH1 methylated primer positive (p = 0.002). 44% (14 from 32) showed positive CDH1 methylation and diagnosed as H. pylori negative (Table 3). From this study, the relationships between CDH1 methylation and participants age showed significance association (P-value 0.025), while gender, clinical symptoms and endoscopy findings were not have statistical significance (All P-value <0.05) (Table 4).

Table 3.

CDH1 Methylation* H. pylori glmM PCR Results Cross Tabulation

		glmM PCR results					p-value
		Positive		Negative		Total
		F	%	F	%
CDH1 Methylation	Positive	16	89	14	44	30	0.002
	Negative	2	11	18	56	20
Total		18	100	32	100	50

Table 4.

Methylation of CDH1 and Age Group, Gender, Symptoms, Endoscopy Results Cross Tabulation

		CDH1 Methylation				P-value
		Positive		Negative
		F	%	F	%
Age/Years	20 - 35 years	9	30	8	40	0.025
	36 - 50 years	2	7	7	35
	51 - 65 years	12	40	3	15
	66 - 80 years	7	23	2	10
	Total	30	100	20	100
Gender	Male	17	57	10	50	0.431
	Female	13	43	10	50
	Total	30	100	20	100
clinical symptoms	Gastric reflex	9		7		0.472
	Epigastric Pain	22	73	16		0.425
	lack of appetite	11	37	8		0.522
	Dysphagia	3	10	1		0.472
	Nausea	11	37	5		0.291
	Vomiting	14	47	8		0.432
	Blood vomiting	3	10	3		0.456
	Chest pain	6	20	4		0.645
	Black feces	8	27	5		0.582
Endoscopy findings	Normal	1	3	2	10	0.769
	Gastritis	18	60	13	65
	Eosophagitis	8	27	3	15
	Duodenal ulcer	1	3	1	5
	Stomach cancer	2	6	1	5
	Total	30	100	20	100

Discussion

Helicobacter pylori infection causes stomach cancer through numbers of pathogenic pathways. In particular, several driver genes are influenced by aberrant DNA methylation (Such as p16, CDH1, hMLH1 and RHOA) which inactivate them. Even noncancerous stomach mucosae can have aberrant DNA methylation, creating an “epigenetic field for cancerization” (Maeda et al., 2016).

In the present study we analyzed 50 samples from clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy to investigate the methylation status of CDH1 gene using MSP technique and correlated it with H. pylori infection. H. pylori was detected in 36% (18/50) of study population using glmM gene PCR test, 89% (16/18) of H. pylori positive cases were CDH1 methylated primer positive (chi-square, p-value=0.002). This suggested strong association between H. pylori infection and CDH1 methylation. This data is consistent with Chan et al., (2006) who reported this association for the first time in 2003. Various results about this association have been reported. The finding of Kague et al., (2010)’s group demonstrated a positive association between H. pylori and CDH1 methylation. Maekita et al., (2006) and other researchers indicated that H. pylori infection potently induces methylation of CpG Island to various degrees.

In addition, Niwa et al., (2013)’s group studied Prevention of Helicobacter pylori-induced gastric cancers in gerbils, their results showed that, elimination of H. pylori clearly resulted in a decrease in methylation levels and a reduction in histological inflammatory responses. Our results disagree with other studies demonstrated that, Epithelial CDH1 expression is significantly reduced in H. pylori infected gastric tissues (Aguilar et al., 2001; Tahara et al., 2009). The techniques used to detect the prevalence of H. pylori infection were very difference, these may justify these contradiction.

Findings from MSP technique showed that, the unmethylated primer was positive in 20% (10/50) of study cases and negative in 80% (40/50) of study case. Appling cross tabulation analysis between methylated and unmethylated CDH1 primers results: 26% (13/50) of study cases showed negative results with both primers. In this study no techniques were used to check the complete bisulfite conversion was occur and excluded the possibility that successful amplification could be attributable to incomplete bisulfite conversion. Kague et al., (2010) used Bisulfite genomic sequencing of the representative PCR products of CDH1 gene and showed that all cytosine at non-CpG sites were completely converted to thymine.

Other findings from MSP technique showed that, 6% (3/50) of study cases have band with both primers. Many researchers suggested the use of real-time methylation specific-PCR when methylation level was calculated as the fraction of methylated molecules in the total number of DNA molecules (Vaissie et al., 2009; Ando et al., 2008; Kikuyama et al., 2012).

Although there was no statistical significant correlation between CDH1 methylation positive gastric mucosa and endoscopy finding (p-value = 0.76). The current study found that DNA methylation can even be present in cancerous and noncancerous gastric mucosa, 60% (18/30) of CDH1 methylation positive gastric mucosa showed gastritis as an endoscopy finding, 27%(8/30) showed eosophagitis, 3%(1/30) showed duodenal ulcer and gastric cancer in 6% (2/30). This supported by the study of Kikuyama et al., (2012) discovered that, diverse expression levels of genes like p16, CDH1, and MLH1 are found in the gastric mucosa, and they are methylated in cancer cells and chronic inflammation. Recently, a multicenter prospective cohort study for predicting the risk of metachronous gastric cancer firstly proved the usefulness of an epigenetic cancer risk marker (Maeda et al., 2016).

In view of epidemiological results among study participants, the age group 20 - 35 years showed higher percentage 34%, while 40% of CDH1 positive cases have an age group 51 - 65 years and there was a significant correlation between and CDH1 methylation positive results and age group p-value = 0.02. There was no significant correlation between and CDH1 methylation positive results and gender p-value=0.431. These findings agree with Yu et al., (2012) who found no correlations between CDH1 methylation in preoperative peritoneal washes from Gastric Cancer patients and clinicopathologic parameters (all P > 0.05).

Although most study population suffering from Epigastric pain (76%) there was no significant association between clinical symptoms and CDH1 positive methylation (all P-value > 0.05). Wide range of clinical symptoms and low prevalence of CDH1 positive methylation among study population may justifies this insignificance. Several researches discovered that, the DNA methylation can even be present in noncancerous gastric mucosa, and its levels are influenced by H. pylori infection this is related to an increase in inflammatory mediators in the stomach mucosa, including IL-8, GRO, MIP-1, ENA-78, and MCP-1. And this is responsible for most clinical symptoms (Maeda et al., 2016).

In conclusion, CDH1 gene methylation is associated with H. pylori infection in gastric mucosa specimens and it was indicated that H. pylori infection potently induces methylation of CpG islands. CDH1 methylation is common occurrences in samples with chronic gastritis and gastric cancer and the correlation between H. pylori infection and CDH1 inactivation involved in early gastric tumorigenesis.

Author Contribution Statement

All authors contributed equally in this study.