# Epigenetic promoter methylation of hmlh1 gene in human gut malignancies: A comparative study

Arif A Bhat, Hilal A Wani, Mushtaq A Beigh, Showkat A Bhat, Showkat Jeelani, Akbar Massood, Sabhiya Majid

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Arif A Bhat, Hilal A Wani, Mushtaq A Beigh, Showkat A Bhat, Showkat Jeelani, et al.. Epigenetic promoter methylation of hmlh1 gene in human gut malignancies: A comparative study. Journal of Investigational Biochemistry, 2013, 2, 10.5455/jib.20130409124009. hal-04371768

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Original Research

Epigenetic promoter methylation of hmlh1 gene in human
gut malignancies: A comparative study

Arif A. Bhat<fnref n="1" />, Hilal A. Wani<fnref n="1" />, Mushtaq A. Beigh<fnref n="2" />, Showkat A. Bhat<fnref n="3" />, Showkat Jeelani<fnref n="4" />,
Akbar Massood<fnref n="5" />, Sabhiya Majid<fnref n="1" />

Received: February 27, 2013

Accepted: April 09, 2013

Published Online: April 18, 2013

DOI: 10.5455/jib.20130409124009

**Corresponding Author:**
Dr. Sabhiya Majid,
Prof. & Head
Department of Biochemistry,
Government Medical College (GMC) Srinagar
Jammu & Kashmir India 190010
sabumajid@yahoo.com

**Key words:** Gastrointestinal, Jammu and
Kashmir, Hypermethylation, hmlh1, MS-PCR

Abstract

Gastrointestinal malignancies (GIT) are a major health hazard globally. The incidence of upper GIT malignancies varies widely based on geographic location, race, and feeding habits etc. GI cancers are most prevalent in Jammu and Kashmir region of India and have multi-factorial etiology involving dietary habits, genetic factors, and gene environmental interactions. Cancer development and progression is dictated by series of alterations in genes such as tumour suppressor genes, DNA repair genes, oncogenes and others. Inactivation of the hmlh1 gene expression by aberrant promoter hypermethylation plays an important role in the progression of various cancers. The present study was a hospital based comparative case-control study and the aim was to analyse the promoter hypermethylation of CpG islands of hmlh1 gene in G cancer patients of ethnic Kashmiri origin. Methylation-specific polymerase chain reaction (MS-PCR) was used for the analysis of the promoter methylation status of hmlh1 gene. DNA was extracted from all the samples and was modified using bisulphite modification kit. The epigenetic analysis revealed that frequency of Promoter region hypermethylation of hmlh1 gene among gastrointestinal malignancies was found to be higher in colorectal cancer (60%) followed by oesophageal (50%) and then gastric cancer (46%) and statically the association of promoter region hypermethylation with GIT malignancies was found to be significant (p<0.05). The promoter hypermethylation of the hmlh1 gene was found to be more prevalent in respective male cases of all the three GIT malignancies in a statistically significant manner (p<0.05).

© 2013 GESDAV

INTRODUCTION

GIT malignancies account for about 20% of all cancers
worldwide with gastric and oesophageal cancer topping
the list [1]. Kashmir has higher incidence of GI cancers
owing to peculiar feeding habits and life style.
Colorectal cancer in Kashmir valley is the third most
common GIT cancer after oesophageal and gastric
cancer [2]. The annual incidence of oesophageal cancer
in Kashmir is reported as 42 and 27 for men and
women, respectively per 100,000 individuals [3]. The
development of GIT malignancy is a result of multiple
independent processes that add up to give a malignant

phenotype and include mutations in various different
proto-oncogenes, tumour suppressor genes, and
epigenetic changes in DNA [4, 5]. In neoplasms,
including GIT cancers, epigenetic changes play a key
role in the process of tumorigenesis [6, 7]. Epigenetic
changes mean the changes in the DNA that modify
gene expression without changing its sequence. More
specifically, epigenetics is the study of collective
interaction of multiple mechanisms in establishing
states of chromatin structure, histone modification,
transcriptional activity, and DNA methylation.
Evidence accumulated in recent years clearly

<footnote n="1">¹Department of Biochemistry, Government Medical College (GMC), Srinagar</footnote>

<footnote n="2">²Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar</footnote>

<footnote n="3">³Division of Biochemistry, FVSc and AH, SKUAST-K, Shuhama, Srinagar</footnote>

<footnote n="4">⁴Department of Surgery, Government Medical College (GMC), Srinagar</footnote>

<footnote n="5">⁵Department of Biochemistry, University of Kashmir, Hazratbal, Srinagar</footnote>
demonstrates that epigenetic abnormalities are
important factors in the etiology of virtually all human
cancer types [8-10]. DNA methylation is the most
widely studied epigenetic abnormality in
tumorogenesis. It refers to methylation of cytosine at
CpG dinucleotides. CpG dinucleotides are not
randomly distributed throughout the human genome.
CpG-rich regions, known as CpG islands, are usually
unmethylated in normal cells (with the exception of
imprinted genes and several genes located on
chromosome X) and are typically found associated with
5' promoter end and first exon of numerous genes [11].
It is estimated that there are about 29000 CpG islands
within the human genome. Approximately 50-60% of
all genes contain CpG islands. Alterations of cytosine
methylation are prevalent in human sporadic cancers
[12, 13]. Methylation defects include genome
hypomethylation (resulting in epigenetic activation of
oncogenes and retro elements) and localized aberrant
hypermethylation of CpG islands, resulting in
transcriptional repression of many important genes
[14]. Promoter hypermethylation patterns in human
cancer show strong specificity with respect to the tissue
of origin, and can be found early in tumorogenesis.
Tumour suppressor genes that undergo aberrant
methylation in multiple tumour types may virtually
belong to all cellular pathways, thus aberrant promoter
hypermethylation has relevant consequences for
carcinogenesis. Moreover, the methylation status of an
individual gene promoter may be used for assessing the
prediction, general prognosis, and response to therapy,
underlying the importance of studies on specific
patterns of promoter hypermethylation in tumours [15].
In GI cancers, epigenetic analysis has revealed a
number of genes and pathways inactivated by
methylation-associated silencing [16, 17]. Human
MutL homologue or *hmlh1* is a member of the
mismatch repair system whose function is to replicate
the genome faithfully [18]. Methylation changes in
mismatch repair (MMR) genes affect their function and
result in accumulation of damage leading to genomic
instability [19, 20]. The MMR system maintains
genomic integrity by correcting replicative errors.
Generally, it is accepted that defects in MMR genes are
responsible for the microsatellite instability (MSI)
observed in different diseases including cancer. Point
mutations within the MMR genes seem to be
infrequent; however, promoter hypermethylation has
been suggested as the main cause of MMR gene
silencing [21, 22]. Deficiencies in mismatch repair
(MMR) system result in mutation rates about 100 fold
greater than observed in normal cells [23, 24].
Methylation of the *hmlh1* gene has been correlated with
the loss of protein expression in many cancers of
humans [25].

This study deals with GIT malignancies predominantly

in rural and socioeconomic disadvantaged
population of all regions of Kashmir and focuses on the
analysis of aberrant promoter hypermethylation of CpG
islands of *hmlh1* gene in GI cancers, with special
emphasis on similarities and differences of promoter
hypermethylation of this mismatch repair gene with
respect to the tissue of origin. There are various
controversial reports of its association with
gastrointestinal cancers [26, 27]. The study also
emphasized recent advances in promoter
hypermethylation profiling in regard to prediction and
general prognosis in GI cancers. The Study was a case
control comparative study in which a candidate gene
approach was used to study a key cancer gene (*hmlh1*)
undergoing epigenetic change in gastrointestinal
cancers. The Study was undertaken to understand the
etiology of gastrointestinal cancers in the population of
Kashmiri origin. Also, association of promoter
hypermethylation of *hmlh1* gene with gastrointestinal
cancers and its relation to clinico-pathological
parameters like age and gender was evaluated.

## MATERIALS AND METHODS

**Collection of tissue samples:** The study included 300 surgically/endoscopically obtained gastrointestinal (Gastric, Colorectal and Oesophageal) samples among which 180 were cases (60 from each group) and 120 (40 from each group) were normal gastrointestinal samples. The carcinoma and control samples were obtained in sterilized plastic vials (50 ml volume) containing 10 ml of normal saline from the Department of Surgery and Endoscopic Section of Shri Maharaja Hari Singh (S.M.H.S) hospital, an associated hospital of Government Medical College Srinagar and were stored at -80°C. A part of each sample was sent to histopathology laboratory of S.M.H.S hospital for histopathological confirmation. The information regarding the age and gender for each sample was collected from the histopathological reports and is shown in Table 1.

**Extraction and Modification (Bisulfite treatment) of genomic DNA:** For the isolation of genomic DNA, kit based method was used. The kit used was Quick-g DNA™ MiniPrep supplied by ZYMO RESEARCH. The DNA eluted was stored at 4°C for a short time and at -20°C for longer duration storage. The integrity of the genomic DNA was examined using 1 % agarose gel. The quantity and quality of the isolated DNA was determined by measuring optical density at 260 nm and 280 nm by double beam spectrophotometer (Evolution 60S from Thermo Scientific). DNA was modified by kit based method and the kit used was EZ DNA Methylation™ Kit supplied by ZYMO RESEARCH. DNA modification (i.e. sodium bisulfite treatment) converted unmethylated cytosines to Uracil and hence
enabled to distinguish between the methylated and non-methylated cytosine residues. The modified DNA was then stored at -20°C for further analysis.

**Methyl specific polymerase chain reaction (MSP):**
The methylation status of the *hmlh1* gene was analysed by using methylation-specific PCR (MSP) approach [28]. The bisulfite-treated DNA was used as a template for PCR. Two sets of primers described previously (39-42) one specific for methylated and the other specific for unmethylated version of the gene were used. All unmethylated cytosines in the unmethylated product were converted to thymines after bisulfite treatment and PCR amplification, suggesting that the *hmlh1* gene is unmethylated. However, the cytosines in the CpG dinucleotides of methylated product remained unchanged, as methylated cytosines are resistant to bisulfite treatment, which indicated that the CpG islands of the gene are methylated. The primers used for amplification along with PCR-annealing temperatures, PCR product sizes are shown in Table 2.

For MSP, the total reaction volume was 25 µl containing 50–100 ng of bisulfite modified DNA, 20 pmol of each primer, 25 mM dNTPs, 1 U Taq Polymerase, 2.5 µl 1X PCR buffer (Bangalore Genei, Bangalore). PCR reactions were started by denaturation at 95°C for 5 min, followed by 30 cycles of 94°C for 30s, 60°C (for unmethylated *hmlh1*) and 58°C (for methylated *hmlh1*) for 30s and 72°C for 30s with a final extension at 72°C for 4 min. DNA from normal lymphocytes was used as negative control for methylated alleles of *hmlh1*, and placental DNA treated in vitro with SsiI methyltransferase (New England Biolabs, Beverly, MA, USA) was used as positive control for methylated alleles of *hmlh1*. 15 µl of PCR products were analyzed by electrophoresis on a 3% agarose gel and visualized by ethidium bromide staining.

**Statistical analysis:** The $\chi^2$-test with Odds ratio was used to examine the differences in the distribution of *hmlh1* gene promoter hypermethylation and non hypermethylation between cases and controls of three different GIT malignancies and Fishers exact test was used in case of studying the male and female groups. Odds ratios with 95% CIs were computed using

unconditional logistic regression using Graph Pad Prism Software Version 5.0 by Graph Pad Software 2236, Avenida de la Playa, La Jolla, CA 92037, USA.

## RESULTS

Analysis of promoter hypermethylation of *hmlh1* gene was carried out in 180 invasive primary gastrointestinal cancer cases and 120 normal samples. Among the cases, more than half were amplified with methylated primer thus were methylated. However, few cases were amplified with both methylated and unmethylated primers which can be probably explained by the presence of infiltrating lymphocytes and/or non-malignant epithelial cells in the primary tumours or by the presence of hemi-methylated DNA. 52.2% of the gastrointestinal cancer cases showed bands in methylated (M) wells which confirmed that promoter region of mismatch repair gene (*hmlh1*) in these cases was hypermethylated. Fig. 1 shows MS-PCR results of few representative GIT cancer cases. However in 47.8% of the gastrointestinal cancer cases, bands were visible in unmethylated wells which confirmed that promoter region of *hmlh1* gene in these cases was not methylated. Among histopathologically confirmed gastrointestinal normal samples, 85.8% of the samples were found to be unmethylated and 14.2% were found to be methylated. Fig. 2 shows MS-PCR results of few representative GIT normal samples. Statistically the association of promoter hypermethylation of mismatch repair gene *hmlh1* with gastrointestinal cancer was evaluated by $\chi^2$-test and was found to be significant (p=0.0001). Frequency of Promoter hypermethylation of *hmlh1* gene in gastrointestinal cancers was found to be higher in colorectal (60%) followed by oesophageal (50%) and then gastric (46%) cancer (Table 3 and Fig. 3). Also, the frequency of promoter hypermethylation of *hmlh1* gene was found to be higher in respective male cases of all GIT malignancies and its association with respective GIT malignancies was found to be significant (Table 4 and Fig. 4). However, among females the frequency of promoter hypermethylation was found to be lower and the association of promoter hypermethylation with respective GIT malignancies was found to be statistically insignificant (Table 5).

<table><caption>**Table 1.** Number of Cases and Controls in Gastrointestinal Cancers</caption><thead><tr><th rowspan="2">Type of Cancer</th><th colspan="3">Case</th><th colspan="3">Control</th></tr><tr><th>Male</th><th>Female</th><th>Total</th><th>Male</th><th>Female</th><th>Total</th></tr></thead><tbody><tr><td>Gastric</td><td>35</td><td>25</td><td>60</td><td>25</td><td>15</td><td>40</td></tr><tr><td>Esophageal</td><td>43</td><td>17</td><td>60</td><td>27</td><td>13</td><td>40</td></tr><tr><td>Colorectal</td><td>40</td><td>20</td><td>60</td><td>25</td><td>15</td><td>40</td></tr></tbody></table>
<table><caption>**Table 2.** Shows primers used along with their annealing temperatures and product sizes</caption><thead><tr><th>Nature of Primer</th><th>Sequence (5' - 3')</th><th>Annealing Temp(°C)</th><th>Size (bp)</th></tr></thead><tbody><tr><td rowspan="2">Unmethylated</td><td>F 5'-TTTTGATGTAGATGTTTTATTAGGGTTGT-3'</td><td rowspan="2">60</td><td rowspan="2">115</td></tr><tr><td>R 5'-ACCACCTCATCATACTACCCACA-3'</td></tr><tr><td rowspan="2">Methylated</td><td>F 5'-ACGTAGACGTTTTATTAGGGTCGC-3'</td><td rowspan="2">58</td><td rowspan="2">110</td></tr><tr><td>R 5'-CCTCATCGTAACTACCCGCG-3'</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Figure 1.** MS-PCR results of few representative gastrointestinal malignant cases</figcaption></figure>

Lane 1 represents 50 Bp ladder

Lane 2 represents positive control amplified with methylated primer

Lane 3 and 4 represents gastric cancer case (GC) amplified with methylated primer only

Lane 5 and 6 represents colorectal cancer case (CC) amplified with methylated primer only

Lane 7 and 8 represents oesophageal cancer case (EC) amplified with methylated primer only

<figure><img src="image_3.png" /><figcaption>**Figure 2.** MS-PCR results of few representative gastrointestinal normal samples</figcaption></figure>

Lane 1 represents 50 Bp ladder

Lane 2 represents negative control amplified with unmethylated primer

Lane 3 and 4 represents normal gastric case (GN) amplified with unmethylated primer only

Lane 5 and 6 represents normal colorectal case (CN) amplified with unmethylated primer only

Lane 7 and 8 represents normal oesophageal case (EC) amplified with unmethylated primer only
<table><caption>**Table 3.** Frequency of *hMLH1* gene promoter region hypermethylation in gastrointestinal malignancies:</caption><thead><tr><th rowspan="2">Type of Cancer</th><th rowspan="2">Parameter</th><th colspan="3">Cases</th><th colspan="3">Control</th></tr><tr><th>M</th><th>F</th><th>T</th><th>M</th><th>F</th><th>T</th></tr></thead><tbody><tr><td rowspan="4">Gastric</td><td>Number</td><td>35</td><td>25</td><td>60</td><td>25</td><td>15</td><td>40</td></tr><tr><td>Hypermethylation</td><td>17</td><td>11</td><td>28</td><td>05</td><td>02</td><td>07</td></tr><tr><td>Frequency (%)</td><td>48</td><td>44</td><td>46</td><td>20</td><td>13</td><td>17.5</td></tr><tr><td>Number</td><td>43</td><td>17</td><td>60</td><td>27</td><td>13</td><td>40</td></tr><tr><td rowspan="4">Esophageal</td><td>Hypermethylation</td><td>22</td><td>08</td><td>30</td><td>04</td><td>02</td><td>06</td></tr><tr><td>Frequency</td><td>51</td><td>47</td><td>50</td><td>14</td><td>15</td><td>15</td></tr><tr><td>Number</td><td>40</td><td>20</td><td>60</td><td>25</td><td>15</td><td>40</td></tr><tr><td>Hypermethylation</td><td>28</td><td>08</td><td>36</td><td>02</td><td>02</td><td>04</td></tr><tr><td>Colorectal</td><td>Hypermethylation</td><td>28</td><td>08</td><td>36</td><td>02</td><td>02</td><td>04</td></tr><tr><td></td><td>Frequency</td><td>70</td><td>40</td><td>60</td><td>08</td><td>13.3</td><td>10</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Frequency of *hMLH1* gene hypermethylation in gastrointestinal malignancies</figcaption></figure>

<table><caption>**Table 4.** Promoter Hypermethylation of Male Cases of GIT malignancies.</caption><thead><tr><th rowspan="2">Type of Cancer</th><th colspan="2">Gastric</th><th colspan="2">Esophageal</th><th colspan="2">Colorectal</th></tr><tr><th>Male Cases</th><th>Male Controls</th><th>Male Cases</th><th>Male Controls</th><th>Male Cases</th><th>Male Controls</th></tr></thead><tbody><tr><td>Total</td><td>35</td><td>25</td><td>43</td><td>27</td><td>40</td><td>25</td></tr><tr><td>Hypermethylation</td><td>17</td><td>05</td><td>22</td><td>04</td><td>28</td><td>02</td></tr><tr><td>Non-Hypermethylation</td><td>18</td><td>20</td><td>21</td><td>23</td><td>12</td><td>23</td></tr><tr><td>p-value</td><td colspan="2">0.0310</td><td colspan="2">0.0024</td><td colspan="2">0.0001</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Frequency of *hMLH1* gene hypermethylation in male gastrointestinal cancer cases</figcaption></figure>
<table><caption>**Table 5.** Promoter Hypermethylation of Female Cases of GIT malignancies</caption><thead><tr><th>Type of cancer</th><th colspan="2">Gastric</th><th colspan="2">Esophageal</th><th colspan="2">Colorectal</th></tr><tr><th>Gender</th><th>Female Cases</th><th>Female Controls</th><th>Female Cases</th><th>Female Controls</th><th>Female Cases</th><th>Female Controls</th></tr></thead><tbody><tr><td>Total</td><td>25</td><td>15</td><td>17</td><td>13</td><td>20</td><td>15</td></tr><tr><td>Hypermethylation</td><td>11</td><td>02</td><td>08</td><td>02</td><td>08</td><td>02</td></tr><tr><td>Non-Hypermethylation</td><td>14</td><td>13</td><td>09</td><td>11</td><td>12</td><td>13</td></tr><tr><td>p-value</td><td colspan="2">0.0801</td><td colspan="2">0.1194</td><td colspan="2">0.130</td></tr></tbody></table>

DISCUSSION

GIT malignancy arises as a result of dysregulated cellular signalling which is in turn dictated by alterations in different proto-oncogenes, tumour suppressor genes and also include epigenetic changes in DNA [4, 5]. In neoplasms, including GI cancers, epigenetic changes play a key role in the process of tumorigenesis [6, 7]. DNA methylation in cancer has become the topic of intense investigation. Promoter hypermethylation is an alternative mechanism of gene inactivation in carcinogenesis [29]. Several studies have suggested that aberrant methylation of the promoter causes transcriptional silencing of some important suppressor genes, such as p16 [30], E-cadherin [31], and von Hippel Lindau (vhl) gene [32], and this has been implicated in the carcinogenic process in many cancers [29]. There are several protective mechanisms that prevent the hypermethylation of the CpG islands. These include active transcription, active demethylation, replication timing, and local chromatin structure preventing access to the DNA methyltransferase [33]. To date, nearly 50% of numerous genes have been found to undergo hypermethylation in cancer. The genes that are susceptible are the genes involved in cell cycle regulation (P16INK4A, p15INK4a, Rb, p14ARF), genes associated with DNA repair (hMLH1, BRCA1, MGMT), apoptosis (DAPK, TMS1), angiogenesis (THBS1, VHL), invasion (CDH1, TIMP3), drug resistance, detoxification, differentiation, and metastasis [34].

The *hmlh1* protein, a mismatch repair enzyme, maintains the fidelity of the genome during cellular proliferation. It has no known enzymatic activity and probably acts as a 'molecular matchmaker', recruiting other DNA-repair proteins to the mismatch repair complex [35]. Dysfunction of a mismatch repair system such as *hmlh1* and *hmsh2* could alter microsatellites, short tandem repetitive sequences [36]. The mismatch repair system is composed of a highly diverse group of proteins that interact with numerous DNA structures during DNA repair and replication [37]. Considering the importance of promoter hypermethylation in inactivation of *hmlh1* which is one of the frequently altered genes in many human cancers, a study was

designed to analyse promoter methylation status of this mismatch repair gene (MMR) and its frequency in various GIT malignancies in an ethnic of Kashmiri origin. To determine the promoter methylation status of mismatch repair (*hmlh1*) gene, methylation specific polymerase chain reaction was performed on a DNA isolated from 180 surgically resected and endoscopically obtained gastrointestinal cancer cases and was compared with that of 120 histopathologically confirmed normal tissues. In the present study MSP was used for analysis of the methylation status of *hmlh1* gene. This method provided significant advantages over previous ones used for assaying methylation. MSP is much more sensitive than Southern analysis, facilitating the detection of low numbers of methylated alleles and the study of DNA from small samples. MSP allows examination of all CpG sites, not just those within sequences recognized by methylation sensitive restriction enzymes.

The frequency of promoter hypermethylation of *hmlh1* gene in gastrointestinal cancers was found to be higher in colorectal cancer (60%) followed by oesophageal cancer (50%) and gastric cancer (46%) where as among normal samples, the frequency was found to be higher in gastric (17.5%) followed by oesophageal (15%) and colorectal (10%) samples respectively. However, on reviewing the literature, the methylation frequency ranged from 8 to 50% for *hmlh1* gene in GIT cancers. The lower methylation profile reported in these earlier studies compared with our study may be due to the techniques employed. The association of promoter hypermethylation with GIT malignancies was found to be significant. The genetic analysis revealed that unlike other high-risk regions, Kashmiri population has a different hypermethylation profile of *hmlh1* gene promoter in males and females. Occurrence of *hmlh1* promoter hypermethylation was found to be unequally distributed in males and females with more frequency in males than in females. Frequency of promoter region hypermethylation of *hmlh1* gene in gastrointestinal cancers was found to be higher in colorectal cancer males (70%) followed by oesophageal cancer males (51%) and gastric cancer males (48%). Gastrointestinal carcinogenesis is a stepwise process of the accumulation of genetic and epigenetic abnormalities.
It is clear that promoter hypermethylation of mismatch repair genes is as important for this multistep process as genetic changes in the progression of these cancers. Our study has supplemented the steadily growing list of genes inactivated by promoter hypermethylation in gastrointestinal cancers. These provide not only new insights into the molecular basis of the diseases but also list of interesting candidate genes for the development of molecular markers which might contribute to the improvement of diagnosis and also prognosis. In addition, the fact that methylation can be reversed *in vitro* and the effect of the demethylating agent 5-aza-2'-deoxycytidine *in vitro* raise hope for new treatment strategies for gastrointestinal cancer patients.

## CONFLICT OF INTEREST

The authors declare that there are no competing interests.

## ACKNOWLEDGEMENTS

Authors acknowledge the support provided by Department of Biochemistry, Govt. Medical College Srinagar and Department of Biochemistry, University of Kashmir. A fellowship in favor of H.A. Wani from CSIR is gratefully acknowledged.

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