Development of DNA methylation markers for sperm, saliva and blood identification using pyrosequencing and qPCR/HRM.

Discrimination of body fluids can provide important information in the investigation of crime scenes. The goal of this project was to identify new sets of tissue specific differentially methylated regions (tDMRs) and develop assays that can be utilized for forensic discrimination of body fluids, in particular sperm, saliva and blood. In this study, a sample set containing semen with sperm, semen without sperm, buccal swabs, saliva (oral fluids), venous blood, menstrual blood, vaginal secretions, and sweat/skin samples were used to develop four assays. Two methods for the analysis of DNA methylation biomarkers were developed in this paper: pyrosequencing and quantitative PCR/high resolution melt (HRM) analysis. Using an epigenome wide association study, two markers, NMUR2 and UBE2U, were found to be specific for sperm, based on the fact that mean DNA methylation levels for semen (containing sperm cells) were significantly lower than mean DNA methylation levels of other body fluids. In addition, one marker (SA-6) was hypermethylated in saliva when compared to other body fluids. The assays developed for NMUR2, UBE2U and SA-6 markers can be applied in forensic tissue identification using both pyrosequencing and HRM analysis. Additionally, a set of CpG sites in the AHRR locus were hypomethylated in blood when compared to other tissues using pyrosequencing. However, this locus was not amenable to HRM analysis. Overall, this work demonstrates the discovery and application of tDMRs for forensic applications.

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing.

The accurate identification of body fluids from crime scenes can aid in the discrimination between criminal and innocent intent. This research aimed to determine if the levels of DNA methylation in the locus PFN3A could be used to discriminate vaginal epithelia from other body fluids. In this work we bisulfite-modified and amplified DNA samples from blood, saliva, semen, and vaginal epithelia using primers for PFN3A. Through pyrosequencing we were able to show that vaginal epithelia present distinct methylation levels when compared to other body fluids. Mixtures of different body fluids present methylation values that correlate with single-source body fluid samples and the primers for PFN3A are specific for primates. This report successfully demonstrated that the analysis of methylation in the PFN3A locus can be used for vaginal epithelia discrimination in forensic samples.

High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

Evaluation of DNA methylation markers and their potential to predict human aging.

We present epigenetic methylation data for two genetic loci, GRIA2, and NPTX2, which were tested for prediction of age from different donors of biofluids. We analyzed 44 saliva samples and 23 blood samples from volunteers with ages ranging from 5 to 72 years. DNA was extracted and bisulfite modified using commercial kits. Specific primers were used for amplification and methylation profiles were determined by pyrosequencing. Methylation data from both markers and their relationship with age were determined using linear regression analysis, which indicates a positive correlation between methylation and age. Older individuals tend to have increased methylation in both markers compared to younger individuals and this trend was more pronounced in the GRIA2 locus when compared to NPTX2. The epigenetic predicted age, calculated using a GRIA2 regression analysis model, was strongly correlated to chronological age (R(2) = 0.801), with an average difference of 6.9 years between estimated and observed ages. When using a NPTX2 regression model, we observed a lower correlation between predicted and chronological age (R(2) = 0.654), with an average difference of 9.2 years. These data indicate these loci can be used as a novel tool for age prediction with potential applications in many areas, including clinical and forensic investigations.

Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing.

The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT1, USP49, DDX4, Hs_INSL6_03, Hs_ZC3H12D_05, and B_SPTB_03 were identified to contain tissue-specific differential methylation. Samples ranging from 9-21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR, and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_SPTB_03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied (p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample.

High-resolution melt analysis of the minisatellite D1S80: a potential forensic screening tool.

High-resolution melt (HRM) analysis of the VNTR region of the human D1S80 locus, a 16-bp repeat minisatellite from approximately 400 to over 700 bp in length, was investigated. A Qiagen Rotor-Gene Q using the Type-it PCR HRM kit was used to acquire HRM curves for 14 single, and 16 biallelic, dsDNA samples. The HRM analysis was applicable over a range of DNA concentrations; however the characteristics of the melt curve did depend on the forward and reverse primer ratio. Despite the large amplicon size and the similarities of the repeat sequences, it was possible to discriminate different genotypes. Heterozygotes were clearly different from the homozygous variants and even small differences in the repeat sequence could be differentiated. However, the melt analysis requires a high-resolution system with temperature resolution of 0.02°C or better in order to sort out differences in these large amplicons of near identical GC content (in this case 56%). HRM analysis of amplicons with large repeat sequences can be used as a means of comparing DNA fragments. Examination of multiple sequences can be used to differentiate DNA samples and demonstrate the potential of HRM analysis as a rapid and inexpensive prescreening technique in forensic applications.

The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.

The goal of this study is to explore the application of epigenetic markers in the identification of biofluids that are commonly found at the crime scene. A series of genetic loci were examined in order to define epigenetic markers that display differential methylation patterns between blood, saliva, semen, and epithelial tissue. Among the different loci tested, we have identified a panel of markers, C20orf117, ZC3H12D, BCAS4, and FGF7, that can be used in the determination of these four tissue types. Since methylation modifications occur at cytosine bases that are immediately followed by guanine bases (CpG sites), methylation levels were measured at CpG sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated CpG-associated cytosine bases to thymine bases. The bisulfite modified DNA was then amplified via nested PCR using a primer set of which one primer was biotin labeled. Biotinylated PCR products were in turn analyzed and the methylation level at each CpG site was quantitated by pyrosequencing. The percent methylation values at each CpG site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC3H12D and FGF7 loci differentiated sperm from blood, saliva, and epithelial cells. The C20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS4. The results of this study demonstrate the applicability of epigenetic markers as a novel tool for the determination of biofluids using bisulfite modification and pyrosequencing.

Mutation at the human D1S80 minisatellite locus.

Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7) mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB) guidelines, we found a male mutation rate of 1.04 × 10(-4) and a female mutation rate of 5.18 × 10(-5) with an overall mutation rate of approximately 7.77 × 10(-5). Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM) and the one-step stepwise mutation model (SMM). In this study, we found that this locus fits into the one-step mutation model (SMM) mechanism in six out of seven instances similar to STR loci.

Investigating SNPs flanking the D1S80 locus in a Tamil population from India.

D1S80 is a 16-bp variable number of tandem repeats minisatellite. We analyzed single nucleotide polymorphisms (SNPs) flanking this locus in a Tamil population. Alleles ranged from 15 through 41 repeats, with alleles 18 and 24 being predominant with frequencies of 31% and 34.5%, respectively, suggesting a bimodal allelic distribution. All the 18-repeat alleles are associated with HinfI(+) and FnuAHI(-) restriction site polymorphisms at the 5' and 3' ends, respectively. Allele 24 is associated with HinfI(-) and Fnu4HI(+). Of the alleles tested, 98.5% have a linkage of two specific SNP polymorphisms. If an allele is positive for HinfI, then it is negative for Fnu4HI, and if an allele is negative for HinfI, it is then positive for Fnu4HI, which demonstrates strong linkage disequilibrium between the two polymorphic SNPs. This suggests that reciprocal crossover is not involved in changes in the number of repeats, as few exchanges are seen in the flanking regions. The repeat allele-SNP association might be involved with the internal structure of the locus micropolymorphisms, possibly a double-strand break hotspot.

Detection and evaluation of DNA methylation markers found at SCGN and KLF14 loci to estimate human age.

Recent developments in the analysis of epigenetic DNA methylation patterns have demonstrated that certain genetic loci show a linear correlation with chronological age. It is the goal of this study to identify a new set of epigenetic methylation markers for the forensic estimation of human age. A total number of 27 CpG sites at three genetic loci, SCGN, DLX5 and KLF14, were examined to evaluate the correlation of their methylation status with age. These sites were evaluated using 72 blood samples and 91 saliva samples collected from volunteers with ages ranging from 5 to 73 years. DNA was bisulfite modified followed by PCR amplification and pyrosequencing to determine the level of DNA methylation at each CpG site. In this study, certain CpG sites in SCGN and KLF14 loci showed methylation levels that were correlated with chronological age, however, the tested CpG sites in DLX5 did not show a correlation with age. Using a 52-saliva sample training set, two age-predictor models were developed by means of a multivariate linear regression analysis for age prediction. The two models performed similarly with a single-locus model explaining 85% of the age variance at a mean absolute deviation of 5.8 years and a dual-locus model explaining 84% of the age variance with a mean absolute deviation of 6.2 years. In the validation set, the mean absolute deviation was measured to be 8.0 years and 7.1 years for the single- and dual-locus model, respectively. Another age predictor model was also developed using a 40-blood sample training set that accounted for 71% of the age variance. This model gave a mean absolute deviation of 6.6 years for the training set and 10.3years for the validation set. The results indicate that specific CpGs in SCGN and KLF14 can be used as potential epigenetic markers to estimate age using saliva and blood specimens. These epigenetic markers could provide important information in cases where the determination of a suspect's age is critical in developing investigative leads.

Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.

Determining the type and origin of body fluids in a forensic investigation can provide important assistance in reconstructing crime scenes. A set of epigenetic markers, ZC3H12D, BCAS4 and cg06379435, have been developed to produce unique and specific patterns of DNA methylation that can be used to identify semen, saliva, and blood, respectively. To ensure the efficacy of these markers, developmental validation studies were performed to determine the conditions and limitations of this new tool for forensic analysis. DNA was extracted from human samples and bisulfite modified using commercial bisulfite modification kits. Specific primers were used to amplify the region of interest and the methylation profile of the CpG sites were determined by pyrosequencing. The percent methylation values at each CpG site were determined in multiple samples and averaged for each tissue type. The versatility of these new markers is presented by showing the results of validation studies on sensitivity, human specificity, stability and mixture resolution. When testing the markers using different organisms, we did obtain positive results for certain non-human primate samples, however, all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10ng, depending on the locus, indicating the importance of primer design and sequence in the assay. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. Simulated case- samples were also tested. In the case of mixtures of different cell types, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the validation studies demonstrate the robustness and effectiveness of this new tool for body fluid identification.

Tissue-Specific DNA Methylation Patterns in Forensic Samples Detected by Pyrosequencing®.

In certain circumstances the outcome of a trial may hinge on the ability of a forensic laboratory to determine the identity of biological stains present at crime scenes. An example of such a situation would be the detection of blood, saliva, vaginal fluid, or other body fluid in a specific location whereby its presence would reinforce the victim's or suspect's version of the events that happened during the commission of a crime. However, current serological methods used for identifying body fluids may lack the sensitivity and specificity to identify these fluids, particularly for trace levels. New procedures using proteomic methods and RNA-based gene expression show promise in addressing this issue; however, concerns about stability and relative levels of gene expression remain. An alternative approach is to utilize patterns of epigenetic DNA methylation. DNA methylation is an epigenetic mechanism that regulates the specificity of genes being expressed or silenced in cells. Regions in the human genome referred to as tissue-specific differentially methylated regions account for unique patterns of DNA methylation that are specific for each cell type. This chapter addresses the application of bisulfite-modified PCR combined with Pyrosequencing(®) to detect tissue-specific DNA methylation patterns and perform trace serological analysis. The quantitative nature and precision available with Pyrosequencing presents major advantages in these studies as it permits detection of and contrast between cells with differential levels of methylation. The procedure can be applied to a variety of biological fluids which may be present at crime scenes.

Y chromosome STR allelic and haplotype diversity in a Rwanda population from East Central Africa.

We have analyzed 17 Y-chromosomal STR loci in a population sample of 69 unrelated male individuals of the Rwanda-Hutu population from East Central Africa using an AmpFlSTR® Yfiler™ PCR amplification kit. A total of 62 unique haplotypes were identified among the 69 individuals studied. The haplotype diversity was found to be 0.9970 for this population. The gene diversity ranged from 0.1130 (DYS392) to 0.7722 (DYS385). Comparison of populations in this study with twenty-five other national and global populations using Principal Co-ordinate Analysis (PCA) and phylogenetic molecular analysis using a genetic distance matrix indicates a delineation of all the African populations from other unrelated populations. The results of population pair-wise Fst p values indicate statistically significant differentiation of the Rwandan population when compared with 25 other global populations including four African populations (p=0.0000). Analysis of Molecular Variance (AMOVA) of the Rwanda population with four other African populations indicated a 93% variance within populations and 7% variance among the five populations. A data base search of the 62 haplotypes yielded only one non-African haplotype match, suggesting these haplotypes are unique to the African continent.

Genetic variation of 15 autosomal microsatellite loci in a Tamil population from Tamil Nadu, Southern India.

The genetic profiles for 15 autosomal microsatellite loci were analyzed in a Tamil population from Southern India to study the genetic diversities and relatedness of this population with other national and global populations. Statistical analyses of the data revealed all loci were within Hardy-Weinberg Equilibrium (HWE) expectations with the exception of the locus D5S818 (p=0.011). A significantly greater inter-individual variation (Fst=99%) observed within the individuals among the four subgroups in this study and low population differentiation (Fst=1%) suggests relative genetic closeness of these four subgroups. This indicates that the populations in the southern region of India might have a common ancestry or probably experienced high gene flow during the period of their coexistence. The Neighbor Joining tree derived from genetic distances of samples from this study and other national and global populations show clustering of all the Indian populations in one branch of the tree while the African and Middle Eastern populations cluster in a separate branch. Principal Co-ordinate Analysis of the genetic distance data show clustering similar to the NJ tree.

Y chromosome STR allelic and haplotype diversity in five ethnic Tamil populations from Tamil Nadu, India.

We have analyzed 17 Y chromosomal STR loci in a population sample of 154 unrelated male individuals of the Tamil ethnic group residing in the state of Tamil Nadu, Southern India using AmpFlSTR(R) Yfiler PCR amplification kit. The population samples consist of the following castes: Kongu Gounder (KOG), Nadar Hindu (NAH), Agamudayar (AGA), Parayar (PAR) and other Tamil individuals (MCT) of mixed castes. A total of 152 unique haplotypes were identified among the 154 individuals studied. The haplotype diversity was found to be 0.9935 or higher for all the five groups. The results of population pairwise Fst p values indicate no statistically significant differentiation between the five populations in this study, but the results were highly significant when compared with 12 other global populations (p<0.05). Comparison of populations in this study with other national and global populations using Principal co-ordinate analysis (PCA) using Rst distance matrix indicates a delineation of all the Indian populations from other unrelated populations.