The Study Of DNA Methylation Of Five Target Sequences Located In Genic And Intergenic Regions For Discriminating Monozygotic Twins

Objective: Monozygotic twins （MZ） develop from a single fertilizedegg，so the genomic DNA sequences of them are identical or very similar. Inthe present, the existing genetic marks applied in forensic practice, such asshort tandem repeat （STR）, singlenucleotide polymophism （SNP） andmitochodrial DNA （mtDNA）, could not distinguish two individuals of MZ,resulting in some cases related with MZ can not be solved. Therefore, how todiscriminate two individuals of MZ is an urgent problem in the field offorensic DNA analysis. DNA methylation is a kind of epigenetic phenomenon,which serves as an important factor for modulating the gene expression.Recently it had been reported that the DNA methylation between twoindividuals of MZ was not all the same. In the previous study, we detected thewhole genome DNA methylation profile of two new born MZ individuals. Theresults indicated that there were2205DNA methylation difference sequencesbetween them, from which, we select113sequences as candidates fordiscriminating MZ pairs.In the present study, we try to further select valuable candidates from the113sequences, to evaluate the discrimination power of them by measuring theDNA methylation of large numbers of twin samples using pyrosequencingtechnology.Methods: We collected blood samples and buccal samples from178pairs of twins and all the participants signed informed consent. Whole genomeDNA was extracted by QIAamp DNA Blood Kit from blood samples and byMouthwash Kit from buccal samples. DNA was genotyped with19STRmarkers using Godeneye DNA Identification System Basic Kit. The genotypeprofiles of two individuals were compared to identify MZ or DZ. The whole genome DNA was performed bisulphite conversion using EZ DNAMethylation-Gold Kit, which was used as template to amplify five targetsequences located in genic or intergenic regions. PCR products weresequenced by pyrosequencing and the quantitative methylation level of3CpGsites was obtained. Data were statistically analyzed using SPSS13.0Results:1Characterization of five target regionsAccording to the MID-Sequence results from one pair of newborn MZ,five differential methylation regions were selected to be analyzed among largesamples of twins. These five regions include three gene region sequences （GS）and two intergenic region sequences （IGS）. Three GSs were respectivelylocated in the exon of transmembrane channel-like4（TMC4），beta-1,4-N-acetyl-galactosaminyl transferase2（B4GALNT2），and proteintyrosine kinase6（PTK6）, while two IGSs were located in Chromosome3（ACCESSION NUMBER: NC₀00003.12）.2Mean methylation level of five target regions in blood samples and buccalsamplesFor total356blood samples the mean DNA methylation levels observedin GS1, GS2, GS3, IGS1and IGS2were88%,89%,92%,85%and82%respectively. Considerable inter-individual variation in DNA methylationvalues was observed within each CpG unit. Within a single amplicon, DNAmethylation values of the CpG units were correlated with each others.To evaluate the tissue specificity, GS1and GS3were also detected in126buccal samples. The results showed that the mean DNA methylation levels ofblood samples for6CpG units in GS1and5CpG units in GS3were92%,83%,74%,97%,93%,89%,93%,98%,90%,90%, and89%respectively.These values are significantly higher than that in buccal samples, for whichthe mean methylation levels were66%,65%,57%,68%,66%,62%,89%,96%,86%,87%, and88%respectively. Among the samples investigated,102blood samples and buccal samples were collected from the same individual.Paired t-test was performed between the two forms of samples from the same individual, and the results further verified the tissue difference of DNAmethylation level between buccal samples and blood samples.3Methylation differences between individuals of MZ pairs or DZ pairsDuo to the sensitivity of methylation analysis by pyrosequencing provesto be5%, we set5%as the criterion to evaluated whether there is differencebetween two individuals of twins. That is to say, if the methylation differencevalue is larger than5%between two individuals, it will be considered that themethylation level of these two individuals is significant different.3.1Methylation differences in blood samples between individuals of MZ pairsor DZ pairsFor blood samples, the number of MZ pairs showed significant differencein GS1, GS2, GS3, IGS1and IGS2was38,0,18,31, and57respectively,which accounted for32.2%,0%,15%,26.72%,49.57%%of the total testedMZ pairs. While that of DZ pairs in the five regions was21,0,5,23, and12respectively, accounting for36.1%,0%8.77%,42.59%,21.82%of the totaltested DZ pairs. When it comes to all of the five regions,93pairs of MZ and39pairs of DZ could be discriminated, accounting for77.5%and67.24%ofthe total detected samples, indicating that the discrimination power of the fiveregions for MZ and DZ was77.5%and67.24%respectively.Then, the discrimination power of five regions on MZ pairs and DZ pairswas compared by χ2test. The results showed that the obvious difference wasonly observed for IGS1and IGS2. For IGS1, the percentage of MZ withmethylation difference larger than5%was significantly less than that of DZ,while for IGS2, the situation is quite the opposite. However, when these twoIGS region was concerned together, the discrimination power on MZ pairs andDZ pairs was not different. For another3GS regions, there was no significantdifference about the discrimination power between MZ pairs and DZ pairseither calculated separately or totally. Overall, if the results of5regions werecombined, no significant difference was obsevered.In addition, the ability of discriminating twins between GS region andIGS region was also evaluated. According to the results, the GS regions, including13CpG units, could discriminate50pairs of MZ and23pairs of DZ,accounting for41.67%and39.66%of the total number of detected samples,while the IGS regions, including7CpG units, could discriminate73pairs ofMZ and29pairs of DZ, accounting for60.83%and50.00%of the totalnumber of detected samples. This indicated that the discrimination power ofIGS regions on MZ pairs was obviously higher than that of GS regions.3.2Methylation differences in buccal samples between individuals of MZpairs or DZ pairsFor buccal samples, the number of MZ pairs showed significant differencein GS1and GS3was40and15respectively, which accounted for80%and76.92%, of the total tested MZ pairs, while that of DZ pairs in the two regionswas10and1respectively, accounting for32.61%and7.69%, of the totaltested DZ pairs. When the two regions was calculated in combination,42pairsof MZ and10pairs of DZ could be discriminated, accounting for84%and76.92%of the total detected samples, which was significantly higher than thatin blood samples detected using the same two regions. This indicated that thediscrimination power for either MZ pairs or DZ pairs in buccal samples washigher than that in blood samples using the same regions.3.3The correlation between methylation difference and ageTo evaluate the effect of age on the methylation differences between twinpairs, the spearman correlation coefficient was calculated between age andmethylation difference values. For IGS1and IGS2regions, weak relationshipbetween age and methylation difference values was observed with r values-0.276and-0.229respectively. However, unexpectively, with the growth ofage, the difference between two individuals of MZ pairs showed a weaktendency of decrease. For two GS regions, no obvious relationship betweenage and methylation difference values was observed.4. Evaluation of age, gender, ethnicity, and smoke influences on DNAmethylation of five regions4.1Effect of age on DNA methylation of five regionsThe blood samples include178pairs of twins with age raging from0to 74years. The samples was divided into six age classes （0-10,11-20,21-30,31-40,41-50,>50）. The methylation level among different age groups showedstatistically significant differences to some extend for all of the five regions inblood samples, especially between younger group and older gourp. Results ofcorrelation analysis also indicated significant either posotive or negativerelationship between age and methylation level of GS1, GS2, IGS1and IGS2in blood samples, and the r values was0.223,-0.214,-0.376,-0.202,respectively.As for buccal samples, significant differences among different age groupswere observed only for GS3region, but not for GS1region. With the increaseof age, the methylation of GS3exhibited a tedency of elevation to somedegree, with r value being0.241.4.2Effect of gender on DNA methylation of five regionsThe volunteers donated blood samples comprise170males and186females, and the buccal samples include44males and82females. Thevariation between females and males was also analyzed. The results suggestedthat either for the5regions in blood samples or the2regions in buccalsamples, there were no significant differences between females and malesgroup, and no obvious relationship bwteen gender and DNA methylation wasobserved.4.3Effect of ethnicity on DNA methylation of five regionsThe volunteers come from six populations including Han, Hani, lahu,zhuang, hui, and Yi. The later four populations were combined to one groupdue to less samples. According to the results of Spearman correlation analysis,only methylation of GS2was related to ethnicity, and no evident correlationwith ethincity was detectable for other4regions. Although differencesbetween Han and another two ethnicity goups were observed for CpG3site ofGS1and CpG5site for GS3, there was no significant difference among3ethnicity groups when concerning the mean methylation level of GS1and GS3.For GS2, regardless of CpG2site or the mean methylation level of the wholesequence, there were evident differences between Han and the other two ethnicity groups.4.4Effect of smoke on DNA methylation of five regionsAmong the five regions, methylation of GS3was related little stronglywith smoke, and another four regions had no relationship with smoke.Although differences between smokers and non-smokers were found for CpG2site of IGS1and CpG1site for IGS2, there was no evident differences for themean methylation levels of IGS1and IGS2. For GS3, significant differencesbetween smokers and non-smokers were observed for CpG5and CpGmean inblood samples, and CpG4site in buccal samples.Conclusions:1) The cummulative discriminating power for MZ pairs of the five targetregions was reached to77.5%. Other than GS2, the rest four regions had thecapacity to distingiush MZ pairs more or less, which might be as the valuablecandidate regions for discriminating MZ pairs. For the intergenic regionsshowed a higher discriminating efficiency than gene regions, suggesting thatintergenic regions might be a better choice to be a candidate region. Besides,for the same sequence, the discrimination power of it in buccal samples wasgreater than that in blood samples, indicating the tissue differences ofmethylation differences between MZ pairs. In addition, with the growth of age,the methylation differences of MZ pairs showed a tendency of decrease, thereason of which is remained to be studied.2) DNA methylation levels of five target regions were all related withage, which suggested that age was an important factor affecting DNAmethylation of nuclear genome, either for genic regions or for intergenicregions. DNA methylation level of B4GALNT2exon was correlated withethnicity, and DNA methylation of PTK6exon was related to smoke,indicating that these two genes are respectively affected by ethnicity andsmoke status.3) As same as our previous study, there was signifcant tissue differencesof DNA methylation for GS1and GS3. DNA methylation values in bloodsamples were greatly higher than that in buccal samples. Therefore, wesuggested that we must use the same kind of tissue sample to do comparisons when it is used in case practice.