Hypomethylation of the DNMT3L Promoter in Ocular
Surface Squamous Neoplasia
Guru Prasad Manderwad, MSc; Gopinathan Gokul, MSc; Chitra Kannabiran, PhD; Santosh G. Honavar, MD;
Sanjeev Khosla, PhD; Geeta K. Vemuganti, MD, DNB, FNAMS
NContext.—Cancer is known to have epigenetic inputs,
with events like genomewide hypomethylation and gene-
specific hypermethylation of DNA. The DNA methyltrans-
ferase enzymes act as effectors of this reprogramming. A
previous study revealed that hypomethylation at the DNA
methyltransferase 3-like (DNMT3L) promoter could be a
potential biomarker in cervical tumors. Because the
pathobiology of ocular surface squamous neoplasia (OSSN)
is similar to that of cervical tumors, we wanted to
determine whether similar changes occur in the methyla-
tion pattern at the DNMT3L promoter in OSSN.
Objective.—To evaluate the methylation status of the
DNMT3L promoter in OSSN compared with healthy
DNMT3L promoter in the tumor tissues of 6 patients with
histologically proven OSSN and in healthy conjunctiva
tissue from 7 individuals for controls using the sodium
bisulfite–assisted conversion of genomic DNA. Extracted
genomic DNA was treated with sodium bisulfite and
amplified with specific primers for the DNMT3L promoter
region. The specific polymerase chain reaction products
were cloned and sequenced.
Results.—The mean age of these patients was 50.2 years
(range, 35–65 years). Histologically, 4 OSSN cases were
invasive; 2 were intraepithelial. Healthy conjunctival tissues
exhibited a methylated promoter region, whereas a variable
loss of methylation was observed in all 6 OSSN cases.
Conclusions.—We have, for the first time to our
knowledge, identified loss of methylation at the DNMT3L
promoter in OSSN cases, but its physiologic significance is
yet to be understood. Further studies are warranted to
substantiate our results.
(Arch Pathol Lab Med. 2010;134:1193–1196)
are squamous in origin and encompass both intraepithe-
lial and invasive tumors involving the limbus, conjuncti-
va, and cornea.1Clinically, OSSN appears as a papilliform,
leukoplakic lesion or a gelatinous mass with superficial
vessels. The etiologic factors implicated in its genesis
include ultraviolet B rays2and high-risk human papillo-
The genetic predisposition to cancer has been well
recognized, but increasing evidence points to the role of
epigenetic mechanisms in the initiation and progression of
cancer. A wide array of epigenetic changes, including
methylation, and aberrant histone modifications, are the
hallmark of most cancers.5–7Both promoter methylation–
cular surface squamous neoplasia (OSSN) is the most
common tumor of the ocular surface. These tumors
mediated silencing of tumor suppressor genes and
activation of oncogenes because of promoter hypomethyl-
ation have been well documented.8–13The DNA methyl-
transferase 3-like (DNMT3L) gene is a member ofthe DNA
methyltransferase 3 (DNMT3) gene family14and regulates
DNA methylation by interacting with the de novo
andDNAmethyltransferase3B (DNMT3B).15,16DNMT3L is
highly expressed in gametes and is a known factor
responsible for the acquisition of imprints during game-
togenesis.17In a previous study, we reported the loss of
methylation at the DNMT3L promoter in cervical cancers.18
Because the pathobiology of OSSN is similar to that of
cervical squamous cell carcinoma, we hypothesized that
OSSN may harbor similar epigenetic changes. Hence, our
promoter of DNMT3L in squamous cell tumors of the
MATERIALS AND METHODS
The present pilot study was approved by the Institutional
Review Board. Fresh tumor tissues (n 5 6) were collected from
patients with a clinical diagnosis of OSSN and stored in liquid
nitrogen. After histologic confirmation of the diagnosis, the
tissues were thawed for DNA extraction. Healthy conjunctival
tissue specimens (n 5 7) were collected from an ongoing project
on characterization of limbal and conjunctival stem cells, which
was also approved by the Institutional Review Board.
Accepted for publication November 24, 2009.
From the Ophthalmic Pathology Services (Mr Manderwad and Dr
Vemuganti), the Kallam Anji Reddy Molecular Genetics Laboratory (Dr
Kannabiran), and the Department of Ophthalmic Plastic Surgery, Orbit
and Ocular Oncology (Dr Honavar), Kallam Anji Reddy Campus, L. V.
Prasad Eye Institute, Hyderabad, India; and the Laboratory of
Mammalian Genetics, Centre for DNA Fingerprinting and Diagnostics,
Tuljaguda, Nampally, Hyderabad (Mr Gokul and Dr Khosla).
The authorshave no other relevant financialinterestin the productsor
companies described in this article.
Reprints: Geeta K. Vemuganti, MD, DNB, FNAMS, Ophthalmic
Pathology Services, HyderabadEye ResearchCentre,KallamAnjiReddy
Campus, L. V. Prasad Eye Institute, L. V. Prasad Marg, Banjara Hills,
Hyderabad 500 034, India (e-mail: email@example.com).
Arch Pathol Lab Med—Vol 134, August 2010Hypomethylation of DNMT3L Promoter in OSSN—Manderwad et al1193
The homogenized tissue samples were suspended in DNA
extraction buffer (100mM Tris—hydrogen chloride at pH 8.0, 50
mM ethylenediaminetetraacetic acid [EDTA], 1% sodium dode-
cyl sulfate, 200 mM sodium chloride, and 100 mg proteinase K)
and incubated overnight at 37uC. Genomic DNA was purified
using the phenol-chloroform extraction method and ethanol
Bisulfite Conversion and Polymerase Chain Reaction
DNA methylation analysis of the DNMT3L promoter was
carried out using the sodium bisulfite–assisted genomic DNA
conversion method described previously by Gokul et al18Briefly,
1 mg of genomic DNA was denatured and mixed with low-
melting agarose (BMA, Rockland, Maine) and pipetted into cold,
heavy mineral oil (Sigma-Aldrich, St Louis, Missouri) to form
beads. These beads were incubated in a bisulfite modification
solution first at 0uC for 30 minutes and then at 50uC for 3.5 hours.
The beads were then incubated with 0.2 M sodium hydroxide
(Sigma-Aldrich) and washed with 13 Tris-EDTA and sterile
deionized water. The polymerase chain reaction (PCR) was
performed using specific primers (hDNMT3LbisF: 59-TTAGTTT-
TATTGAGTTTTTAATTTT-39; hDNMT3LbisR: 59-TAATAAATT-
CAAAATTCCAATAAT-39 [MWG Biotech, Bangalore, India])
amplifying a 375–base pair region within the DNMT3L promoter.
The amplification was performed in a thermal cycler (Eppendorf
Master Cycler, Hamburg, Germany), with initial denaturation at
95uC for 5 minutes, followed by 35 cycles of denaturation at 94uC
for 1 minute, annealing at55uC for 1 minute,and extensionat 72uC
for 1 minute.
Cloning and Sequencing of PCR Products
The PCR products were ligated to a TA-based cloning vector
(designed by inserting 2 Xcm1 sites in the p Blue Script SK+
[Stratagene] vector, which gives T [thymidine] and A [adenosine]
overhang after digestion) and transformed into Escherichia coli
(DH10B [Invitrogen, Carlsbad, California]) competent cells.
Ampicillin-resistant colonies were screened for the presence of
the insert by colony PCR. Plasmids were isolated from positive
colonies and were sequenced using vector-specific primers.
Multiple clones (range, 3–11 per sample) were sequenced to
obtain a complete methylation profile.
The differences in DNA methylation levels at the DNMT3L
promoter in OSSN and in healthy conjunctiva were analyzed
using the t test for each CpG at all loci examined. The t test was
done using 1-tailed distribution and taking into account unequal
variance in the 2 data sets. The differences were considered to be
statistically significant when the P values were less than .05. The
efficacy of the bisulfite conversion was found to be 98.6% (SEM 5
0.56%) for all cytosines converted to thymine in a non-CpG
We studied the methylation levels of 11 CpG dinucle-
otides in the DNMT3L promoter. The mean age of the 6
patients with OSSN included in the study was 50.2 years
(range, 35–65 years); 4 patients were histologically
diagnosed as having invasive tumors, and 2 had intraep-
ithelial tumors. All 6 cases of OSSN showed hypomethyl-
ation at the DNMT3L promoter region as compared with
the control samples, as shown in Figure 1.
Among the clones analyzed, most CpGs were methyl-
ated at an average rate of 60% in controls but at a rate of
39% in OSSN samples (obtained by calculating the
percentage of methylation at each CpG dinucleotide for
each sample and averaging across the control or tumor
samples). The summary of the DNA methylation profiles
for the controls and OSSN are presented in Figure 2. Of
the tumor samples that we analyzed, OSSN4 showed a
complete loss of DNA methylation at all the CpG
dinucleotides except the 11th CpG (3 clones were screened
for OSSN3; however, those 3 clones exhibited a uniform
profile, except for CpG3 in clone 2). In the healthy
conjunctiva tissues, the fourth CpG was found to be
devoid of any methylation, whereas the 11th CpG was
found to be methylated in all the clones (except in few
clones of conjunctiva 3). The methylation profile of the 4th
and 11th CpGs in OSSN cases resembled that of controls
(except for CpG 11 in OSSN1, which was totally
unmethylated). Hence, these CpG dinucleotides might
not be informative with respect to methylation changes in
The statistical significance of methylation differences at
each CpG dinucleotide was determined by comparing
healthy conjunctiva and OSSN samples. We found that the
loss of methylation in OSSN cases was statistically
significant (P , .05) at the third, sixth, and ninth CpGs
Because of the small number of samples available for
each tumor grade, we could not get any statistically
significant correlation with DNA methylation.
The variability observed in the methylation at individ-
ual CpGs, even between the different clones of the sample,
could be attributed to 2 aspects of the technique. Because
each clone represents a profile of the single cell, the
heterogeneity between the cells could be due to difference
in epigenetic states of each cell in a tissue. It is also
possible that during sample collection, some healthy cells
were collected along with the tumor cells, which could
account for the variability in the methylation profile.
Different cell types are characterized by their unique
gene expression patterns, which, in turn, are governed by
a wide array of regulatory mechanisms. There are several
epigenetic processes governing gene regulation.19,20Epi-
genetic modifications, especially DNA methylation, play a
key role in that regulation. DNA methylation in the
promoter region has been well known to bring about the
silencing of the relevant gene.21Although the question
remains as to whether these epigenetic changes are a cause
or a consequence of cancer, many studies have supported
the presence of such changes in various stages of cancer.22
Aberrant DNA methylation is a well-known epigenetic
hallmark in almost all cancers,23including squamous cell
carcinoma of cervix.18Alteration in DNA methyltransfer-
ases plays an important role in the development of
cancer.24Given that the DNMT3L promoter has been
shown to be hypomethylated in cervical cancers, we
evaluated for methylation differences at this locus using a
limited number of OSSN tumors.
Results from this pilot study indicate that varying
degrees of hypomethylation can occur in the DNMT3L
promoter region in OSSN. This loss of methylation could
possibly lead to increased levels of gene expression at the
DNMT3L locus in these tumors. Direct estimation of the
level of expression of DNMT3L would be required to
confirm this; however, that was not possible in this study
because of limitations in the amount of tissue available.
Increased levels of DNMT3L in cancer could lead to
aberrant DNA methylation, and subsequent misregula-
tion of a subset of genes, because of its stimulatory effect
1194Arch Pathol Lab Med—Vol 134, August 2010Hypomethylation of DNMT3L Promoter in OSSN—Manderwad et al
on DNMT3A and DNMT3B.20
methylation differences at the third, sixth, and ninth
CpGs to be statistically significant, the differences at the
sixth CpG were more prominent. We speculate that this
CpG could possibly be a suitable molecular marker, using
a methylation-specific PCR strategy, which could serve as
Although we foundan adjunct screening method for OSSN. However, any use
of this strategy as a molecular diagnosis tool for OSSN
would need to be further validated on a larger sample.
To our knowledge, this study is the first to have shown
epigenetic changes in OSSN. Our data are corroborated
by previous studies showing hypomethylation at the
Methylation profiles for the promoter region of DNMT3L in healthy conjunctiva (CON) and OSSN. Each horizontal line denotes a single clone. The
11 CpG dinucleotides analyzed within the DNMT3L promoter are represented as circles. Filled circles denote a methylated cytosine, whereas open
circles denote an unmethylated cytosine.
Diagrammatic representation of methylation pattern of CpGs in ocular surface squamous neoplasia (OSSN) and control tissues.
methylation in different CpGs in ocular
surface squamous neoplasia and healthy
conjunctiva. Summary of DNA methylation
analysis at the DNMT3L promoter. Each box
represents one CpG dinucleotide. The profile
for all 11 CpG dinucleotides is represented in
rows for each of the specimens tested. Colors
denote the percentage of clones showing
methylation at individual CpG dinucleotides
as follows: green, 0% to 34%; yellow, 34% to
66%; red, 66% to 100%. Abbreviations: CIN,
conjunctival intraepithelial neoplasia; con,
conjunctiva; OSSN, ocular surface squamous
neoplasia; SCC, squamous cell carcinoma.
Representation of the degree of
Arch Pathol Lab Med—Vol 134, August 2010 Hypomethylation of DNMT3L Promoter in OSSN—Manderwad et al 1195
DNMT3L promoter in cervical cancer. As reported for
cervical cancer, we could not determine any specific
correlation between tumor grades in the OSSN cases
examined studied and changes in DNA methylation
because of the small numbers of tissues evaluated. In
conclusion, we observed DNA hypomethylation at the
DNMT3L promoter in OSSN. These observations suggest
that epigenetic events play a role in the genesis of OSSN
and raise the possibility that changes in methylation at this
locus could be a potential biomarker for OSSN. Further
studies to determine methylation and activity of the
DNMT3L gene in a large number of cases are required to
confirm these possibilities.
We thank the Hyderabad Eye Research Foundation and the
Indian Council of Medical Research (ICMR; 5/4/6/13/02-NCD-
II) for funding this research. G.P.M. received his research
fellowship from the ICMR project. G.G. is a senior research
fellow of Council of Scientific and Industrial Research (CSIR).
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Methylation at Each CpG in the DNMT3L Promoter in
Ocular Surface Squamous Neoplasia (OSSN) and
in OSSN, %
aThe significance of difference in methylation at each CpG dinucle-
otide was calculated using the 1-tail distribution t test. The difference at
the third, sixth, and ninth CpG was found to be statistically significant
(P , .05).
1196Arch Pathol Lab Med—Vol 134, August 2010 Hypomethylation of DNMT3L Promoter in OSSN—Manderwad et al