Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification

Journal of Forensic and Legal Medicine 34 (2015) 34e39

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Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification Lijuan Bai a, b, e, Peng Yan a, b, d, e, Ximei Cao b, c, Linna Jia a, b, Ce Zhang b, Dawei Guo a, b, * a

Department of Forensic Science, Shanxi Medical University, Taiyuan, 030001, China Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China c Department of Histology and Embryology, Shanxi Medical University, Taiyuan, 030001, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2015 Received in revised form 5 May 2015 Accepted 7 May 2015 Available online 16 May 2015

Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%. © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Keywords: H19 imprinting control region Sperm DNA Methylation-sensitive restriction enzyme Real time PCR

1. Introduction Human genomic imprinting refers to monoallelic expression caused by parental-specific gene marking. There are at least 90 imprinting genes in human genome. Many imprinting genes form clusters spanning 1 Mb or more. One of the clusters located on chromatin 11p15.5 is H19/Igf2 imprinting cluster. In this cluster H19 gene shows the maternal expression, whereas paternal allele is silenced by means of DNA methylation and histone modification.1 Therefore, the paternal allele-specific methylation resided in a GC-rich region on H19 ICR is one of the characterizations of H19 imprinting markers.2 A pile of documents focused on parent-of-polymorphism in H19 gene ICR reveal to paternal specific allele methylated SNP or VNTR in samples originated from blood, skeletal muscle, skin, cerebrum,

* Corresponding author. Department of Forensic Science, Shanxi Medical University, 56 South Xijian Nan Road, Taiyuan, 030001, Shanxi, China. Tel.: þ86 01186 351 4135147, þ86 01186 13015409491 (mobile). E-mail address: [email protected] (D. Guo). d Present address: Department of Forensic Sciences, Xi'an Jiaotong University, Xi'an, 710061, China. e These two authors contributed equally to this work.

liver etc.3e5 Based on Naito reports,4 four HhaI sites are involved in paternal specific methylation located on human chromosome 11 2004477-2006247 (Supplementary data 1) on H19 ICR. That means only about half amount of DNA originated from somatic cells would be intact in this region after HhaI digestion. Whereas Jing-Yu Li et al. reported that H19 ICR retains both paternal and maternal methylation in mature sperm of mouse.6 We presume that human mature sperm also shows such biallelic methylation status in this region, and thus the HhaI sites either from paternal allele or maternal allele are resistant to HhaI digestion. The differential methylation levels on H19 ICR between sperm and somatic cells prompt us to design an experiment to discriminate sperm DNA and somatic DNA. The existence of semen in forensic samples usually couples to sexual assault crime. Until now there are many approaches to be employed to discover the semen by presumptive tests and confirmatory tests.7 Seminal acid phosphatase test is often used to detect semen as presumptive test because of occurrence of phosphatase activity from semen as well as other tissues. Searching for sperm cells under microscope is a reliable method to identify the semen. However, the examination under microscope is a time-consuming task and needs the skilled personnel to perform. Albeit immunological assay can also be used to detect the seminal specific antigen such as

http://dx.doi.org/10.1016/j.jflm.2015.05.001 1752-928X/© 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

L. Bai et al. / Journal of Forensic and Legal Medicine 34 (2015) 34e39

prostate specific antigen(p30) and semenogenlin(Sg),7,8 protein is less stable than DNA and degradation of protein might cause falsenegative result for aged casework specimen examination. Tissue specific mRNA profiling and miRNA signature are used to trace back to biological stain origin including semen in recent papers.910 But in comparison with DNA, RNA molecules are far more easily degraded because of widely distribution of ribonuclease(RNnas) enzyme in environment.11 Saverio Giapaoli et al.12 reported forFLUID kit testing results from eight laboratories for identification of vaginal fluids by analyzing the commensal bacteria genomes. The multiplex realtime PCR was used to probe genomic DNA from six microbes. The vaginal stains were identified by all eight laboratories with the simple and efficient multiplex real-time PCR. In some sexual abuse criminal cases it is useful evidence to find the vaginal stain on relevant matter of criminal suspect when semen is not discovery. However, sperm identification followed by STR profiling, in most cases, is more favorable evidence for court. Recently, some groups developed new semen identification kits based on differential DNA methylation in human genomic DNA.13e16 With aid of the methylation sensitive restriction enzyme the DNA fragments linked to differential DNA methylation markers are amplified and separated by capillary electrophoresis. Ja Hyun an et al.13 tested four human tissue-specific differentially methylated regions(tDMRs) by MSRE-PCR and methylation SNaPshot. The sperm DNA can be identified from 40 semen samples, and furthermore the menstrual blood and vaginal fluids were dicriminated from blood and saliva samples by these four tDMRs. Adam Wasserstrom et al.14 designed a practical semen proving system with adequate negative and positive control. In their semen identification profile the sperm DNA can be distinguished by MSREPCR-capillary electrophoresis from blood and saliva, the latter two samples are mostly mixed with semen in sexual abuse cases. The method of differential DNA methylation marker testing used by these two groups promises to automotive semen identification and avoids to the disadvantages of traditional semen identification ways. However, multiplex fluorescent primers for PCR and capillary electrophoresis of amplified products make testing kits being expensive. Here we recommend a rather simple and non-expensive approach to detect sperm DNA. Without figuring out the size of DNA fragments, The DNA is subjected to HhaI digestion, followed by two round of PCR. In our preliminary study, sperm DNA can be identified by this simple method. 2. Material and method 2.1. Experimental design Fig. 1A and B shows H19 HhaI sites analyzed in this assay and MSRE-nested-qPCR workflow respectively. 2.2. Sample collection Thirty-two blood samples were collected from male and female volunteers. Semen samples were from 31 healthy male volunteers. Seventeen abnormal semen samples from men suffered from oligoasthenoterazoospermia according to criteria of the World Health Organization17 were gathered up from Second Hospital in Shanxi Medical University. The ways of sample collection were based on the procedures approved by the Ethics Committee of Shanxi Medical University. Collections of samples including 100 blood and 8 semen samples were in the same way as mentioned above. These samples are

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for preliminary test. The sample collection procedure was obeyed the rule of Ethics Committee of Shanxi Medical University. 2.3. DNA extraction and MSRE-nested-PCR The phenol-chloroform extraction protocol was used for DNA extraction from donor samples.18 The composition of digestion and PCR mixture (50 ml) was as follows: two nanogram of human genomic DNA, 1.5 ml Quick Cut™ HhaI (Takara), 1.25 U TaKaRa Ex Taq® HS, 25 ml 2  GC BufferⅠ, 0.2 mM each dNTP, 0.2 mM of A-F and A-R primers and sterile water to a total volume of 50 ml. The combined digestion and first round PCR cycling program was 37  C for 15 min, 65  C for 20 min, 95  C for 5 min and thirtyfive cycles of 94  C for 30s, 63  C for 30s and 72  C for 2 min with last extension at 72  C for 5 min in Life Touch Thermal Cycler (BIOER). The second round of qPCR cocktail contained 1 ml of diluted first round PCR amplicons (1/30), 10 ml 2  SYBR® Premix Ex Taq™ II(TaKaRa),1 ml inner primers (10 pmol/ml each) and 8 ml sterile water. After denaturing 95  C for 30s, the PCR profile for 35 cycles was 95  C for 10s, 55  C for 20s and 72  C for 20s in Real-Time QPCR System Mx3000P(Stratagene). 2.4. Subcloning of DNA fragment in human H19 upstream region (chrom 11 2004477-2006247) All HhaI sites in plasmid DNA isolated from E Coli. can be cut by HhaI because of lacking of CpG MTases in E Coli. Therefore, we made a construct by subcloning DNA fragment in human H19 upstream region (chrom 11 2004477-2006247) into pGL3 plasmid to set up a non-methylation HhaI site control. DNA fragment to be inserted was amplified by PCR using primers sub-clone F and sub-clone R. The PCR products were digested by KpnⅠand XhoⅠand cloned into pGL3 vector (Promega) with KpnⅠand XhoⅠsites. 2.5. Evaluation of MSRE-nested-qPCR amplification bias The DNA fragments in human H19 upstream region from plasmid with or without HhaI digestion were mixed by different ratios and subjected to two round PCR amplification. The ratios of amplicons between HhaI digestion and un-digestion were compared to ratios of DNA fragment mixture to evaluate PCR amplification bias. Primers. Outer primers for MSRE-Nested-qPCR analysis4: A-F: 50 -GGGTCATTATAGACGCAATCG-3' A-R: 50 -AGAACCTGTTGGGCGGTTAGA-3' Inner primers for MSRE-Nested-qPCR analysis: C-F: 50 -CTGGGAACACTGGGAAAG-3' C-R: 50 -AGAAACTGGGCTGATTGG-3' H19 ICR subcloning primers: Sub-clone F: 50 -CAGTGGTACCGGGTCATTATAGACGCAATCG-3’ Sub-clone R: 50 -TACGCTCGAGAGAACCTGTTGGGCGGTTAGA-3' 2.6. Statistic analysis SPSS17.0 software was used to analyze data. We used Shapiro-Wilk test and independent T-test to test the methylation measurement level distribution function and to compare methylation measurement level in blood samples and semen respectively. A cut-off corresponding to average methylation measurement level in sperm DNA minus 2 standard deviation(SD) was used to discriminate sperm DNA which was in biallelic methylation status.

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3. Results 3.1. Differential DNA methylation status between sperms and somatic cells To identify biallelic methylation status in human sperm the VNTR phenotypes on H19 imprinting locus were typed according to Naito et al. reported.4 We chose DNA from semen samples and blood cells to carry out the methylation-sensitive restriction enzyme-PCR(MSRE-PCR). As shown in Fig. 2, the DNA from sperm was not sensitive to HhaI digestion while sample from blood was partly digested by HhaI. One hundred blood samples and 8 semen samples were tested and there were no exception. 3.2. Evaluation of MSRE-nested-qPCR amplification bias

Fig. 2. The different methylation status between sperm and blood cells. The HhaI digestion and PCR protocols were based on Naito et al4, Lane 5 DNA Marker, Lanes 1, 2: individual 1, lanes 3, 4: individual 2, Lanes 6, 7: individual 3; Lanes 1, 2, 3, 4: DNA from sperms. Lanes 6, 7: DNA from blood; Lanes 1, 3, 6: HhaI digestion. Lanes 2, 4, 7: HhaI un-digestion.

We evaluated MSRE-nested-qPCR amplification bias by mixing HhaI digested and HhaI undigested DNA fragments from plasmid to

Fig. 1. The design for semen identification by methylation-sensitive restriction enzymes based-nested-qPCR(MSRE-nested-qPCR). A Diagrammatic sketch for MSRE-nested-qPCR region (Chro 11:2004477-2006247, Supplementary 1). A-F & A-R stand for outer primers for first round PCR. C-F & C-R are inner primers for second round of real-time PCR. B Workflow to discriminate between sperm DNA and DNA from blood samples. Theoretically, DNA methylation level in blood sample is 50%(digested/undigested DNA), while sperm DNA methylation level equals to 100% in H19 ICR.

L. Bai et al. / Journal of Forensic and Legal Medicine 34 (2015) 34e39 Table 1 The results for evaluation of MSRE-Nested-qPCR amplification bias. The ratios of HhaI undigested/digested DNA templates for MSRE-Nested- qPCR

The percentage of HhaI undigested DNA templates for MSRE-nested-qPCR

The percentage of PCR products by MSRE-nested-qPCR

0:1 1:4 1:2 1:1 2:1 4:1 1:0

0 20% 33.30% 50% 66.6% 80% 100%

0.00% 19.62% 32.88% 47.94% 68.70% 78.92% 100%

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Table 2 The average value of DNA methylation level with 95% confidence interval of each group tested by MSRE-Nested-qPCR in H19 gene ICR (Supplementary data 1).

Number of samples Average value 95% confidence interval

Blood samples

Sperm samples

32 55.27a±8.36%b 52.25-58.28%

31 101.94c±11.66% 97.66-106.21%

a DNA methylation level tested by MSRE-Nested-qPCR using HhaI digested/undigested DNA as templates. b Standard deviation. c The cut-off level for sperm DNA discrimination: average DNA methylation level in sperm DNA 2SD ¼ 101.94  2  11.66 ¼ 78.62.

4. Discussion mimic different methylation levels. As shown by Table 1 the ratios of amplicons by MSRE-Nested-qPCR are consistent with ratios of mixed DNA fragments between HhaI digestion and undigestion. As shown in Fig. 3 the goodness of fit for MSRE-Nested-qPCR value between templates and PCR products exhibited the linear correlation. The correlation coefficient was 0.999. 3.3. Detection of sperm DNA by MSRE-nested-qPCR We then tested the H19 DNA methylation levels by comparison MSRE-Nested-qPCR products between HhaI digested and undigested DNA templates in semen and blood samples. The average value of DNA methylation levels in semen and blood samples were 101.94 ± 11.66% and 55.27 ± 8.36% respectively (Table 2). Fig. 4 shows methylation levels for 31 semen samples and 32 blood samples. There is a gap between lowest DNA methylation level (79.12%) in sperm DNA and highest DNA methylation level (69.13%) in DNA from blood samples (Supplement data 2). Statistical analysis by Shapiro-Wilk test demonstrated that the methylation levels were in consistent with normal distribution. DNA methylation status in semen and blood samples was significantly different in H19 ICR (p < 0.01, Fig. 4).

Fig. 3. Linear correlation between percentage of HhaI digested/un-digested templates and PCR products by MSRE-Nested-qPCR. The DNA from HhaI digested/undigested samples were subjected to two round of PCR, A pair of A-F and A-R primers was used for first round PCR, and another pair of primers(B-F and B-R) was used for second round of real time PCR dyed by SYBR. X axis denotes percentage of HhaI digested/ undigested template for MSRE-Nested-qPCR, Y axis denotes percentage of PCR products by MSRE-Nested-qPCR. The correlation coefficient (R2) is 0.999.

In mammal somatic cells H19 imprinting control region (ICR) is in specific paternal allelic methylation throughout the lifetime of individuals.19 However, in mature sperm the H19 ICR is undergone the de novo methylation at both paternal and maternal alleles on male germ cells due to genomic wide epigenetic re-programming.20.21,22,23 The phenomena suggest that H19 ICR methyltion level is 50% lower in somatic cells than that in sperm DNA. We examined methylation status of H19 ICR reported by Naito et al.4 in human sperms and somatic cells. The results showed the biallelic methylation in sperms (Fig. 1). Further experimental data suggested that at least three HhaI sites in this region were in unmethylated status in blood samples. whereas no unmethylated HhaI site was found in sperm DNA using T-linker PCR24 (Data not shown). In our design whatever which one is in unmethylated status among these four HhaI sites in the region of chrom11 2004477-2006247 the first round of PCR amplification will be blocked. Therefore, sperm DNA and somatic DNA can be distinguished by comparison of ratio between digested and undigested genomic DNA, which reflects the DNA methylation status. To ascertain whether we are able to know the methylation status by MSRE-nested-qPCR without obvious amplification bias we mixed the HhaI digested/undigested H19 ICR DNA originated from plasmids with different ratio and amplified DNA by MSRE-nested-qPCR. The linear correlation for ratio between MSRE-Nested-qPCR products and DNA template mixture is 0.999, which means the H19 ICR methylation status can be precisely probed by MSRE-nested-qPCR. The average DNA methylation level from 31 sperm and 32 blood samples as Table 2 shows are consistent to the notion that there is biallelic methylation on H19 ICR in sperm and monoallelic methylation occurs in blood sample. Compared to highest 69.13% methylation level from 32 blood samples, the lowest sperm DNA methylation level is 79.12% among 31 semen samples (Supplementary data 2). So, there is a methylation level gap between semen and blood samples. This implies us that DNA from sperm and somatic cell could be discriminated by HhaI site methylation level. The cut-off level of sperm DNA methylation was above 78.62%, which was from equation as Table 2 shows.25 However, based on our data the lower methylation level should not be attributed to DNA from somatic DNA solely. For example, the sperm DNAs from oligoasthenoterazoospermia patients express low H19 ICR methylation level (Supplementary data 2). In this case we are unable to tell the difference between somatic DNA and sperm. Another reason is, when sperm as a minor component mixed with somatic cells the sperm DNA might be hidden and the mixed sample appears low DNA methylation level, which mainly reflect major component somatic cells’ methylation level. In these circumstances, microscopic testing for sperm is necessary. In forensic lab, real time PCR is frequently used for template quantification. The MSRE-nested real time PCR is a potential

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Fig. 4. Different methlyation level between sperm(C) and blood(:). Methlytion level in H19 gene upstream region (Supplementary data 1) from 32 blood and 31 semen samples expressed by Scatter diagram (A) and Box plot (B). The distribution of methylation level in both samples is consistent with normal distribution by Shapiro-Wilk test. The DNA methylation level of semen and blood samples was significantly different by T test (t ¼ 18, P < 0.01).

approach to quantify DNA template and identify semen simultaneously after the multiplex TaqMan technique introduced. So, there are still have rooms to optimized the MSRE-nested-real time PCR. In our experimental design, the size of first round PCR amplicon is 1.7 kb. Using such a large amplicon has an inherent risk to produce false negative signal in fragmented DNA which is frequently observed in forensic casework samples. Thus the present protocol needs to be improved and to be further evaluated before application in casework.

5. Conclusion Differential DNA methylation on human H19 ICR in sperm and blood could be probed by MSRE-nested-qPCR. The sperm DNA can be identified if the DNA methylation level is above 78.62%.

Conflict of interest Author's conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article.

Funding This work was supported by National Science Foundation of China (Grant No. 90608008), Natural Science Foundation of Shanxi Province, China (Grant No. 200601110), Ph.D. programs foundation of the Ministry of Education of China (Grant No. 20111417110003).

Ethical approval None.

Acknowledgments We thank scientific research & experimental center of Shanxi medical university for providing equipments.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jflm.2015.05.001.

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