Research paperLINE-1 DNA methylation: A potential forensic marker for discriminating monozygotic twins
Introduction
At present, paternity testing and individual identification in forensic casework can be resolved by testing a set of 16–24 short tandem repeat (STR) genetic markers [1]. However, criminal or paternity cases with monozygotic twins (MZ) as suspects, or as alleged fathers cannot be resolved using such technology, since MZ twins develop from a single fertilized egg and share the identical gene sequence which cannot be distinguished by DNA sequence analysis. Therefore, discriminating two individuals of MZ twins is an urgent problem in the field of forensic DNA analysis. Although there is increasing evidence of genetic differences, such as copy number variations (CNVs) [2], [3], [4], single nucleotide polymorphisms (SNPs) [5], and satellite DNA [6] within MZ twin pairs it has been concluded that genetic discordance is not common within MZ twin pairs [2]. Therefore, establishing routine tests using genetic discordance is not practical.
Another possible method to distinguishing MZ twin pairs may be epigenetic markers. It has been confirmed that epigenetic differences contribute to the significant phenotypic discordances between MZ twins [7]. Several studies have reported that MZ twin pairs showed obvious epigenetic differences including DNA methylation and histone modification, and both whole genome DNA methylation and locus-specific DNA methylation were found to be different between MZ twins [8], [9], [10], [11], [12], [13]. Therefore, differences in 5-methylcytosine (5mC) within MZ twins pairs may be an appropriate biomarker for discriminating MZ twins.
In our previous study, we studied whole genome DNA methylation profiles of two newborn MZ individuals using methylated DNA immunoprecipitation (MeDIP) sequencing technology. The results indicated that there were 2205 significant DNA methylation difference sequences between them, most located in interspersed repeat sequence such as Alu and long interspersed element (LINE-1) [14]. There may be two reasons: (i) the selective pressure is relatively low for the repeat sequences, so the genetic variation and epigenetic variation is relatively greater than other sequences; (ii) the copy number of LINE-1 and Alu sequence are so high that they account for the most part of the MeDIP sequences. LINE-1 elements are present at greater than 50 million copies, accounting for 17% of the human genome. Therefore the methylation level of LINE-1 can be representative of the methylation level of the whole nuclear genome [15], [16]. To further evaluate the discrimination power of LINE-1 DNA methylation for discriminating MZ twins, this study investigated the LINE-1 DNA methylation of a large number of twins.
Section snippets
Samples
We collected peripheral blood samples in K2 EDTA anticoagulant tubes and buccal samples by sample collection swabs (Tailang Biotechnology, China) from 176 pairs of healthy twins including 84 pairs of male twins and 92 pairs of female twins, and all the participants signed informed consent. The volunteers come from three populations including Han (109 pairs), Hani (52 pairs), and Yi (15 pairs). The volunteers of Han population are living in Hebei, Shanxi and Yunnan province, while the residence
Sample statistics
The demographic characteristics of the population studied are shown in Table 1.
Mean methylation levels and correlation among three CpG sites
The average methylation levels of three CpG sites among 176 unrelated twin pairs were 81.26% ± 2.1%, 73.91% ± 1.8%, and 74.74% ± 2.4% in blood samples, and the mean methylation degree of three sites was 76.60% ± 1.5% (Fig. 2). LINE-1 methylation level of three CpG sites averaged 73.18% ± 5.2%, 69.86% ± 3.4%, and 67.85% ± 4.7%, respectively among 64 individuals in buccal samples, and the mean methylation degree of three sites was
Discussion
This study examined the methylation level of three CpG sites located in LINE-1 promoter region of 176 pairs of healthy twins. The average methylation levels of the three CpG sites in the blood samples were 81.26%, 73.91%, 74.74% respectively, and the mean methylation level of 3 CpG sites was 76.60%. Bollati et al. [17] reported that LINE-1 methylation level average 83.1% in 38 cases of healthy individuals from Milan, Italy detected by pyrosequencing. Using the same method, Burris et al. [18]
Author contributions
SL and BC designed the study. JX, GF and LF recruited twins and collected samples. JX, GF and XZ extracted DNA, bisulphite converted and performed pyroseqencing. LY and CM carried out the statistic analysis. All authors contributed to interpretation of the data. SL and JC wrote the first and last drafts of the manuscript and all authors made critical revisions. SL and BC take responsibility for the integrity of the data and accuracy of the data analysis.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by the National Natural Science Foundation of China [30973364]; and Hebei Province Outstanding Youth Science Fund [H2012206103].
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