Forensic Science International: Genetics Supplement Series 3 (2011) e337–e338

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/FSIGSS

Identical but not the same: The value of DNA methylation profiling in
forensic discrimination within monozygotic twins
Chengtao Li, Suhua Zhang, Tingzhi Que, Li Li, Shumin Zhao *
Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Sciences, Ministry of Justice, 1347 West Guangfu Road, Shanghai 200063, PR China

A R T I C L E I N F O A B S T R A C T

Article history: Monozygotic twins (MZs) show remarkable resemblance in many aspects including behavior and health,
Received 23 August 2011 because they share identical genomic DNA. However, evidence for epigenetic differences within MZs has
Received in revised form 6 September 2011 been accumulated. DNA methylation differences between MZs could partially account for their
Accepted 15 September 2011
phenotypic discordance of behavioral traits and diseases. High throughput epigenomic microarray
profiling can be a strategy of choice for identification of epigenetic differences in phenotypically different
Keywords: MZs. In this study, we mapped MZs DNA methylation differences in white blood cells by interrogation of
Forensic genetics
the unmethylated genome on methylation Beadchip. Blood samples were taken from 22 pairs of adult
Monozygotic twins
Individual identification
MZs. Genomic DNA was bisulfite modified by EZ DNA methylation-Gold kit according to the
DNA methylation manufacturer’s protocols, consequently analyzed with Illumina’s Human Methylation27 Beadchip
including more than 27,000 CpG sites. The results indicated that MZs exhibited remarkable differences
among their genome-wide 5-methylcytosine. According to a set of selection criteria, 377 CpG sites with
significant differences of methylation status were picked out. Although DNA methylation shows only
partial stability, primary results of this study strongly suggested that the CpG methylation could be a
perspective biomarker to distinguish MZs from each other.
ß 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2. Materials and methods

It has been always difficult to perform individual identification
within monozygotic twins (MZs) in some forensic circumstance. It
is impossible to distinguish MZs from each other with conven-
tional DNA genetic markers, such as short tandem repeats (STRs)
and single nucleotide polymorphisms (SNPs). However, MZs nearly
always show various degrees of phenotypic discordance. In the last
decade, evidences have been accumulating that epigenetic
modifications of DNA and histones can have a primary role in
phenotypic outcomes, including human diseases [1,2]. These
results provided an unprecedented idea to how to discriminate
MZs for forensic purpose. For the application perspective of
forensic genetics, DNA methylation may be one of the most
promising epigenetic markers in the discrimination of MZs. In this
study, an epigenome-wide scanning was employed to select those
potential CpG sites for discrimination of MZs.
* Corresponding author. Tel.: +86 021 52352959; fax: +86 021 52352959.
E-mail address: zhaoshuminxl@hotmail.com (S. Zhao).
2.1. Sample collection
Blood samples were taken from 13 pairs of female and 9 pairs of
male MZs, respectively. Range of age of the MZs is from 17 to 74
years old. Written informed consent was obtained from all
participants, and the study was approved by the local ethics
committee. Genomic DNA was extracted using a QIAamp DNA
Blood Mini Kit according to manufacturer’s instructions (Qiagen,
Hilden, Germany).
2.2. Determination of monozygosity
All the genomic DNA samples of 22 pairs of MZs were
genotyped with AmpFlSTR Identifiler kit and an inhouse inser-
tion/deletion polymorphisms (InDel) genotyping system [3,4] to
determinate their monozygosities. The amplified products were
separated by capillary electrophoresis on 3130 Genetic Analyzers,
data of which were analyzed together with an allelic ladder and
positive and negative controls using GeneMapper ID Software
Version 3.2.

1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2011.09.031
e338 C. Li et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e337–e338
2.3. Bisulfite treatment
Genomic DNA was bisulfite modified by EZ DNA methylation-
Gold kit (Zymo Research, Orange, CA, USA) according to the
manufacturer’s suggested protocols for the Infinium Methylation
Assay. Bisulfite sodium converts unmethylated cytosine to uracil.
The bisulfate-modified DNA was stored at 20 8C and used within
a week for analysis.
2.4. Statistical analysis
Background normalization was conducted using the negative
control signals from each sample. Average normalization was done
to minimize chip-to-chip variation. The average intensity values of
the first color channel for all the wells in each chip were used to
calculate mean value, which was scaled to 1. At the end of this
procedure, each chip has the same average intensity in each color
channel. Methylation status for each CpG site is showed as a beta
(b)-value, ranging from 0 (completely unmethylated) to 1
(completely methylated), which was definite as a ratio of
methylated-probe signal intensity to total signal intensity of
methylated and unmethylated probes).
3. Results and discussion
STR and InDel profiles of each sample were determined and
compared to the corresponding siblings. When no differences were
detected in the 15 STR loci provided by the Identifiler Kit and in the
30 InDel markers provided by InDel_typer30 system, the twins
were considered as being MZs. In all cases, the true MZ twin status
was confirmed.
In this study, differences of DNA methylation profiles of each
pair of the 22 MZs were illustrated with volcano plots according to
the criteria of ABDs  13 and ABDb  0.17. These volcano plots had
revealed the CpG sites with differential methylation status within
each pair of the 22 MZs. Consequently, 377 candidate CpG sites
with differential methylation status was determined, each of
which meet the two criteria in at least one pair of MZs.
This initial work presented here indicates the potential value of
an especial kind of epigenetic marker, CpG methylation, in
distinguishing MZs from each other. Furthermore, a definite
candidate pool of CpG sites was also determined in our study.
However, it still has a long way to go to utilize the primary results
in forensic caseworks, because major results in our study will need
to be replicated in larger cohorts of MZs and some key issues must
be scientifically clarified including the metastability of CpG
methylation, tissue or population specific conservation of methyl-
ation status of those candidate CpG sites, development of reliable
and feasible methylation detection system for multiple CpG sites.
Role of funding
This study was supported by grants from the National Nature
Science Foundation, PR China (no. 30901701 and 81172908),
Nature Science Foundation of Shanghai (no. 11ZR1438400).
Conflict of interest
None.
Acknowledgement
The authors would like to thank Dr. Jinzhong Chen for reviewing
the manuscript.
References
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