| Objective:Monozygotic twins (MZ) develop from a single fertilized egg, so the genomic DNA sequences of them are identical or very similar. In the present, the existing genetic marks applied in forensic practice could not distinguish two individuals of MZ, resulting in some cases related with MZ can not be solved. Therefore, how to discriminate two individuals of MZ is an urgent problem in the field of forensic DNA analysis.5-methylcytosine (5mC) i.e., at the5carbon atom of cytosine to add a methyl group, is an important epigenetic phenomenon, which is called the sixth kind of base. Recently it had been reported that there were differences of5mC between MZ two individuals. In the previous study, we detected the whole genome DNA methylation profile of two new born MZ individuals. The results indicated that there were2205DNA methylation difference sequences between them, most located in interspersed repeat sequence such as Alu and long interspersed element (LINE-1). The copies of LINE-1are greater than50million, accounting for17%of the human genome, therefore the methylation level of LINE-1can be representative of the methylation level of the whole nuclear genome. To further evaluate the discrimination power of LINE-1DNA methylation for discriminating MZ, this study investigated the LINE-1DNA methylation of a large number of twins.Methods:We collected blood samples and buccal samples from176pairs of twins and all the participants signed informed consent. Whole genome DNA was extracted by QIAamp DNA Blood Kit from blood samples and by Mouthwash Kit from buccal samples. DNA was genotyped with19STR markers using Godeneye DNA Identification System Basic Kit. The genotype profiles of two individuals were compared to identify MZ or DZ. The whole genome DNA was performed bisulphite conversion using EZ DNA Methylation-Gold Kit, which was used as template to amplify target LINE-1sequence. PCR products were sequenced by pyrosequencing and the quantitative methylation level of3CpG sites was obtained. Data were statistically analyzed using SPSS13.0Results:1Sample statistics176pairs of blood samples include119pairs of MZ and57pairs of DZ.64pairs of buccal samples include50pairs of MZ and14pairs of DZ.2Mean methylation levels and correlation among three CpG sitesThe average methylation levels of three CpG sites among352individuals were respectively81.26%,73.91%, and74.74%in blood samples. However, they were73.18%,69.86%, and67.85%among128individuals in buccal samples, which were significantly lower than that in blood samples, indicating the tissue differences of LINE-1DNA methylation. Among the samples investigated,110blood samples and buccal samples were collected from the same individual. Paired t-test was performed between the two kinds of samples from the same individual, and the results further verified that the methylation level in buccal samples was significantly lower than that in blood. The DNA methylation values of the3CpG sites were strongly correlated with each other according to the correlation coefficient matrix analysis.3Methylation differences between MZ and DZ pairsPaired t-test was done between two individuals of each pair of twins to verify the true methylation difference between them. Among119pairs of MZ,15pairs could be discriminated according to the difference of CpG methylation level between them, accounted for12.61%of total number of MZ. As for DZ,10pairs had significant difference between two individuals, accounted for17.54%of the total56pairs of DZ. To evaluate the effect of age on the methylation differences between twin pairs, the spearman correlation coefficient was calculated between age and methylation difference values. Regardless of MZ or DZ, no obvious relationship between age and methylation difference values was found, indicating that the methylation difference of LINE-1between MZ or DZ was not increase with age. In addition, the degree of methylation difference was compared between MZ and DZ, and no significant difference was observed.4Evaluation of age and gender influences on LINE-1methylationThe blood samples include176pairs of twins with age raging from0to74years. The samples was divided into six age classes (0-10,11-20,21-30,31-40,41-50,>50). Because the methylation values of3CpG sites have strong correlation between each other, the data are consistent with principle components analysis (PCA). Therefore, PC1values and CpGmean, i.e. the mean value of3CpG sites were used to analyze the relationship with age. No statistically significant correlation was detected between age and PC1/CpGmean values. The Kruskal Wallis test also confirmed that no statistically significant difference was observed among different age groups. However, significant positive relationship was observed between age and LINE-1methylation in buccal samples, which was also confirmed by Kruskal Wallis test.The volunteers donated blood samples comprise168males and184females, and the buccal samples include44males and84females. The variation between females and males was also analyzed. The results suggested that in blood samples, regardless of the mean methylation values and CpGmean of PC1, it was lower in females than in males. But no significant difference was found between females and males in buccal samples.5Evaluation of ethnicity and living place influences on LINE-1methylationThe volunteers come from three populations including Han, Hani, and Yi. The volunteers of Han population are living in Hebei and Shanxi. The residence area of Hani and Yi volunteers is Yunnan. According to the results of Spearman correlation analysis and Kruskal Wallis test, no evident differences were detectable among different ethnicity and living places.6Evaluation of profession and education influences on LINE-1methylation To evaluate the effect of occupation on DNA methylation, the samples were divided to manual workers (88blood samples,37buccal samples), mental workers (31blood samples,10buccal samples), minors (68blood samples,30buccal samples) and others (21blood samples,11buccal samples). The Kruskal Wallis test results suggested that regardless of blood samples or buccal samples, both PC1values and CpGmean values showed statistically significant variation among different professions.To further investigate whether educational level affect DNA methylation, the samples were divided to four groups including master degree of higher (5blood samples,0buccal samples), university degree (16blood samples,7buccal samples), high school degree (16blood samples,10buccal samples), primary school degree of lower (136blood samples,73buccal samples). Evident differences were observed in blood samples but not in buccal samples.7Evaluation of smoking influence on LINE-1methylationIn blood samples and buccal samples, no statistically significant variations were detectable between smokers and non-smokers, suggesting that smoking was not a influence factor for LINE-1methylation.Conclusions:In conclusion, this study developed a quantitative method to analyze the CpG methylation level of LINE-1using pyrosequencing technology, which could discriminate two individuals of MZ, and the discrimination power reached to12.61%. Although the discrimination power was not high, this method at least provides an initial feasible solution to solve the forensic MZ discrimination problems. More sequences and sites will be studied in further research to improve the cumulative discrimination power. In addition, due to the tissue differences and age relationship of LINE-1methylation, we must pay attention to the tissue types and age of the samples in practical application. |
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