Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Forensic Science International: Genetics Supplement Series

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

Genome-wide copy number variation analysis in monozygotic twins

⁎
Xiling Liu1, , Zhenmin Zhao1, Qiannan Xu, Zheng Wang, Yingnan Bian, Suhua Zhang,
⁎
Chengtao Li
Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Institute of Forensic Sciences, Ministry of Justice, Shanghai, 200063, PR China

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

Keywords: Increasing evidence indicates the presence of copy number variations (CNVs) between pairs of monozygotic
Monozygotic twins (MZ) twins, especially when they are phenotypically discordant. These results suggest that a genome-wide
Copy number variations comparison of CNV profiles between MZ twins might provide insight into forensic discrimination of MZ twins.
Genome-wide Here, using a high-resolution microarray, we compared the CNV profiles in blood samples collected from MZ
Microarray
twins. We detected 1345 CNV loci. Among them, 31 were shared in at least half of the samples. This study
suggests the potential utility of CNVs in the identification of individual MZ twins.

1. Introduction 2.2. DNA preparation and STR typing

Monozygotic (MZ) twins have long been assumed to be genetically
identical as they are derived from the same zygote, which makes it
impossible to distinguish one from the other using forensic DNA typing
systems. In fact, recent studies have suggested MZ twins can accumulate
somatic mutations after the zygote splits. The most studied type of
structural variant, copy number variations (CNVs), are DNA segments
ranging in size from thousands to millions of bases that differ in the
number of copies across different members of the species [1–5]. CNV
has been reported in MZ twins and suggested as an explanation for
phenotypic discordance [6,7]. These results indicated that a genome-
wide comparison of CNV profiles between MZ twins might provide an
insight into forensic discrimination of MZ twins. Here, using a high-
resolution microarray, we compared the CNV profiles in blood samples
collected from 12 pairs of MZ twins.
2. Methods
2.1. Sample collection
Genomic DNA was extracted from the blood samples using the
QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s
protocol. Homozygosity was confirmed by DNA analysis of STR poly-
morphisms using AmpFlSTR Identifiler Plus Kit (Thermo Scientific).
2.3. Microarray
The genome-wide CNV profiles were measured using the Agilent
SurePrint G3 human CGH Microarray (Agilent Technologies) according
to the manufacturer’s protocol. Each pair was hybridized to one array,
and one was labeled with the Cy3 NHS ester dye and the other with the
Cy5 NHS ester dye. Images of hybridized arrays were acquired using an
Agilent Microarray Scanner (Agilent Technologies). Images were
quantified and normalized with the algorithm LOWESS, using Feature
Extraction software 10.7 (Agilent Technologies). Normalized data were
analyzed further using DNA Analytics 4.0.81 (Agilent Technologies) to
assure the quality of hybridization and to detect aberrations under the
window size = 1 M and the Z-score threshold = 4.0.

Twelve pairs of MZ twins aged 22–47 years were recruited for this
study (6 female pairs and 6 male pairs). All participants were Han
Chinese and had no significant health problems. All individuals pro-
vided informed consent in writing for the use of blood samples. This
study was approved by the Ethical Committee of the Institute of
Forensic Science, Ministry of Justice, P.R. China.
3. Results and discussion
Following an effective and unbiased approach, we detected 1345
loci showing CNV between MZ twins (Fig. 1A). Among them, 24.3%
(N = 327) showed that CN discordance was shared by at least two
pairs, and 2.3% (N = 31) was shared by at least half of the sets of twins
(Fig. 2B and C). More than 62.8% (N = 845) of the CNVs were located

⁎
Corresponding authors.
E-mail addresses: xlliu323@gmail.com (X. Liu), 2550038357@qq.com (Q. Xu), wangzhengtim@163.com (Z. Wang), lichengtaohla@163.com (C. Li).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.fsigss.2017.09.075
Received 28 August 2017; Accepted 15 September 2017
1875-1768/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Liu, X., Forensic Science International: Genetics Supplement Series (2017),
http://dx.doi.org/10.1016/j.fsigss.2017.09.075
X. Liu et al. Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx

Fig. 1. CN discordance between MZ twins. (A) A chromosomal karyogram depicting loci with CN discordance between MZ twins (Black bars are all detected CNVs; red bars are CNVs in
non-coding regions). (B) Overlap between CNV loci in pairs of MZ twins. (C) Overlap between non-coding CNV loci in pairs of MZ twins. (D) Proportions of CNVs located in promoters
(blue), UTRs (green), exons (orange), introns (purple), and intergenic regions (brown). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

in the promoter regions, defined as +/− 1 kb of the transcript start site
(TSS), while 22.7% (N = 305) of the CNVs were located in non-coding
regions, including introns and intergenic regions (Fig. 1C). To test the
general validity of our results, we determined whether there was any
consistent change in the DNA methylation level by using the published
whole-genome methylation data measured in 10 of the same 12 sets of
twins [8]. We found that the changes in the level of DNA methylation
between MZ twins at CNV loci were higher than those at loci with CN
concordance (one-sided Wilcoxon test, P < 0.001 for all CNVs;
P < 0.05 for CNVs located in non-coding regions) (Fig. 2).
4. Conclusion
In this study, we detected 1345 loci showing CN discordance be-
tween MZ twins. We further found the loci with CN discordance showed
more changes in DNA methylation between MZ twins. This pilot study
not only provided a genome-wide level scanning of CN discordance
between MZ twins but also suggested the potential utility of CNVs in the
identification of individual MZ twins.
Conflict of interest

The authors have declared that they have no conflict of interest.

2
X. Liu et al.
Fig. 2. Difference in DNA methylation between MZ twins in non-coding regions showing
CN discordance between MZ twins (red bar), all detected regions showing CN discordance
between MZ twins (orange bar), and regions showing CN concordance between MZ twins
(gray bar). The symbols above the bars show the significance of comparison of DNA
methylation change between MZ twins in different groups based on a one-sided Wilcoxon
test (***: P < 0.001; *: P < 0.05).
Acknowledgments
This study was supported by grants from the National Natural
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Science Foundation of China (grant numbers 81330073 and 81625013),
the Science and Technology Commission of Shanghai Municipality
(grant numbers 17DZ2203800 and 16DZ1205500), the Ministry of
Finance of China (grant number GY2017G-3).
References
[1] R. Redon, S. Ishikawa, K.R. Fitch, et al., Global variation in copy number in the
human genome, Nature 444 (2006) 444–454.
[2] R. Khaja, J. Zhang, J.R. MacDonald, et al., Genome assembly comparison identifies
structural variants in the human genome, Nat. Genet. 38 (2006) 1413–1418.
[3] A.J. Sharp, D.P. Locke, S.D. McGrath, et al., Segmental duplications and copy-
number variation in the human genome, Am. J. Hum. Genet. 77 (2005) 78–88.
[4] L. Feuk, A.R. Carson, S.W. Scherer, Structural variation in the human genome, Nat.
Rev. Genet. 7 (2006) 85–97.
[5] A.J. Iafrate, L. Feuk, M.N. Rivera, et al., Detection of large-scale variation in the
human genome, Nat. Genet. 36 (2004) 949–951.
[6] C.E. Bruder, A. Piotrowski, A.A. Gijsbers, et al., Phenotypically concordant and
discordant monozygotic twins display different DNA copy-number-variation profiles,
Am. J. Hum. Genet. 82 (2008) 763–771.
[7] A. Abdellaoui, E.A. Ehli, J.J. Hottenga, et al., CNV Concordance in 1,097 MZ twin
pairs, Twin Res. Hum. Genet. 18 (2015) 1–12.
[8] N. Zhang, S. Zhao, S.H. Zhang, et al., Intra-Monozygotic twin pair discordance and
longitudinal variation of whole-Genome scale DNA methylation in adults, PLoS One
10 (2015) e0135022.