Abstract
Ever since the discovery of DNA fingerprinting by Sir Alec Jeffreys [1] using human myoglobin gene based probes, the technology has undergone considerable refinement and automation for forensic applications in human, plants of economic importance, extinct animals, human pathogens etc. [2-6]). Forensic epigenetics has lately been gaining significant interest given its emerging value of epigenetic variation in resolving forensically relevant questions. [7] Methylation of DNA has a broad range of effect on the lifestyle, health status and physical appearance of the individual. DNA methylation profiling of forensic trace is useful in the determination of the cell or tissue type of DNA source and also for age estimation of the sample source. The quality and quantity of biosample available from crime scene limits the possible number of DNA methylation test and also selection of the technology that can be used. A wide range of DNA methylation analysis techniques has been used for forensic epigenetic investigations of forensic biological samples. However, development of novel techniques is needed for multiplex analysis of epigenetic markers as the currently available techniques for epigenetic marker analysis requires a large amount of high-quality DNA and also limited in their multiplexing capacities that are often insufficient to fully resolve a forensic query of interest.
1. Introduction
Epigenetic modifications are one of the most important heritable alterations in the DNA. Epigenetic process does not change the DNA sequence, it involves the DNA base modifications such as cytosine 5′-CpG-3′ methylation and post-translational histone modifications such as histone H3 methylation or acetylation. [8] It is involved in regulation of gene expression as a response to various brief or prolonged environmental cues.[9] The methylated CpG islands near gene promoter make it compact and unaccessible to transcription factors leading to inactivation of the gene.[10] Epigenetics has a wide role in human development and is a key regulator of genomic imprinting and X-chromosome inactivation. Mis-programming of epigenetic regulation and aberrant DNA methylation of key regulatory genes affects several human diseases. DNA methylation aberrations have been implicated in various forms of cancer and also in numerous developmental disorders such as Fragile X syndrome,[11] Prader–Willi and Angelman syndrome[12] and Beckwith–Wiedemann syndrome.[13]
Epigenetic is also relevant in the forensic field, the DNA methylation levels adjusted across the genome as a response to various brief or prolonged environmental stimuli result in individual epigenomic variation referred to as epigenetic fingerprint.[14] Compared to other epigenetic modifications such as chromatin structure changes or histone modifications, DNA methylation is preferred in forensics for both in vitro stability and high sensitivity in terms of DNA amounts required. In forensic investigations, differential DNA methylation profile have been analysed for tissue/cell-type identificaton,[15] sex determination,[16] estimation of age[17] and also to differentiate between monozygotic twins.[18] In this minireview, we overview the importance of DNA methylation in forensic studies, the requirements and limiting factor of forensic epigenetic techniques for DNA methylation profiling analysis.
2. DNA Methylation in Forensics
Methylation of DNA has a broad range of effect on the individual lifestyle, health status, physical appearance.[19] It can also reveal information related to one’s socio-economic status, diet, physical activity, alcohol,[20] drug intake and smoking status.[21] For the purpose of identifying the source of a biological trace left at a crime scene, DNA methylation levels are finding use for age estimation[17] and to make predictions about the visible characteristics (such as hair, skin, and eye color),[22, 23] to narrow down the pool of the suspect related to a crime.
Innovative methods have been developed by forensic epigeneticist for profiling and analysis of DNA methylation. However, there are challenges given the quantitative outcome of epigenetic analysis.
3. Requirements for Forensic Epigenetic Analysis
The limiting factor in the progress of forensic genetics and also of forensic epigenetics is the amount and quality of DNA recovered from crime scene. The availability of multiple epigenetic markers and convenient technology for multiplex analysis of the large number of markers is also critical for forensic epigenetic advancement.
3.1. Epigenetic Biomarkers for Forensic Tissue Identification
For forensic trace analysis there is a requirement of cell or tissue specific multiple DNA methylation markers. The amount and quality of biological sample is very limited at crime scene which also restricts the number of DNA test that are possible. Analysis of every single epigenetic marker is time consuming and requires more DNA. In forensic analysis, there is a need for more advance methods for multiplex genotyping for the immediate simultaneous analysis of multiple epigenetic markers.
3.2. Quality Quantity of the Forensic Trace:
Different DNA methylation analysis methods demand large amounts of high-quality DNA whereas forensic biological samples available at crime scenes are limited. Currently available technologies such as DNA methylation microarrays and whole-genome bisulfite sequencing, requires a large amount of high-quality DNA.[24] More advance technologies for the epigenetic marker analysis that are able to deal with small amount or low-quality DNA, such as pyrosequencing and EPITYPER®, are limited in their potential of multiplexing.[25] The amount and quality of DNA recovered from crime scene traces is one of the important key to resolve a forensic question of interest.
3.3. Sample Source Cell/Tissue Type
The identification of cell or tissue type using DNA methylation profile analysis serves as a useful information for crime scene investigations.[26] Several multiplex test assays have been developed for identification and validation of tissue specific epigenetic marker analysis. Forat et al. [27]validated a set of nine DNA methylation markers for the identification of blood, saliva, semen, vaginal fluid and menstrual blood. Using Illumina 450 K database, Lin et al. [28]identified a set of eight tissue-specific epigenetic markers. Similarly, Vidaki et al. identified two novel semen-specific CpG markers (cg04382920 and cg11768416).[29]
4. Techniques for Forensic Epigenetics Analysis
The currently available technologies demand high grade quality DNA for the analysis of a large number of epigenetic markers for forensic trace analysis. These are described below:
4.1. DNA Methylation Microarrays
DNA methylation microarray is a high throughput screening method that utilizes methylation-sensitive restriction enzymes to profile methylated fragments and interrogated them to a CpG island microarray.[30] DNA methylation microarrays offer cost-effective and consistent analysis of many biologically relevant genomic regions. Hong et al, [31] used small sample size (n = 54) and identified six age-associated CpGs in saliva based on the Illumina 450 K microarray platform. For the identification of blood, saliva, semen, vaginal fluid and menstrual blood, Forat et al., [27]identified 150 candidate tissue specific markers based on Illumina HumanMethylation450 Beadchip microarray data.
4.2. DNA Methylation Bisulfite Genomic Sequencing
DNA methylation Bisulfite genomic sequencing is regarded as a gold-standard technology for efficient detection of 5-methylcytosine at single base-pair resolution. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, 5mCs are immune to this conversion. The converted cytosine is recognized as thymine in subsequent PCR amplification and sequencing. All the remaining cytosine residues in the sequence represent previously methylated cytosines in the genome.[32] To produce the methylation maps of a single PCR amplicon traditional method of bisulfite sequencing relied on Sanger sequencing but the advancement in the next-generation sequencing techniques has enabled sequencing of the entire bisulfite-treated genomes and is much faster and cheaper.
4.3. Bisulfite Pyrosequencing
Bisulfite pyrosequencing, another simple and easily available technique for forensic laboratories, also involves the bisulfite conversion of the DNA strand, using specific primers amplification of the target gene region, and real-time DNA sequencing based on sequencing-by-synthesis technology and detection of the release of inorganic pyrophosphate during nucleotide incorporation. Due to its single CpG resolution, highly quantitative nature and sensitivity this technique is by far the most popular for forensic epigenetic analysis.[33] This method has been successfully applied for the analysis of small genomic regions (50-100bps) that can contain multiple adjacent CpG sites. Initial developmental validation studies have shown that single-plex bisulfite pyrosequencing assays are very sensitive (down to 50–100 pg of DNA input, depending on the locus), applicable to old stains and simulated case samples.[34]
4.4. Methylation Quantitative PCR
Methylation quantitative PCR are highly sensitive and quantitative assay to study the methylation level of entire PCR fragments that may contain several CpGs.[35] Real-time qPCR methods is one of the commonly used methods in forensics for the quantitative and qualitative assessment of human-specific genomic DNA. SYBRTM-Green-based qPCR, is an epigenetic qPCR assay which targets one or a few CpG sites in the primer binding region and carried out as single-plex reactions.[36] Whereas, another forensic epigenetic qPCR assay, TaqManTM probe-based assays use different fluorescent dyes and can be multiplexed.[37]
4.5. EPITYPER Mass Spectrometry
The Agena BioscienceMassArray EpiTYPER is a mass spectrometry-based method for region-specific bisulfite sequencing to assess DNA methylation of multiple CpGs in genomic regions of 100–600 base-pairs in a quantitative, single-nucleotide resolution and high-throughput manner.[38] The technology has gained popularity among forensic epigenetic protocols. Using EpiTYPER technology, Freire-Aradas et al.), analyzed DNA methylation levels in a total of 177 CpGs in twenty-two candidate genomic DNA in 725 European individuals and built a novel age prediction model.[39]
4.6. Methylation SNaPshot
The Applied Biosystems SNaPshot technology, also uses treatment of genomic DNA with sodium bisulfite to discriminate unmethylated from methylated cytosines to generate a DNA template for quantitative methylation analysis.[40] Strand-specific PCR is performed using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for rapid quantitation of methylation at individual CpG sites. For visualization the reaction products are electrophoresed on polyacrylamide gels and quantitated by phosphorimager analysis. This method requires small amounts of DNA and in a single primer extension reaction the methylation status of several CpG sites can be determined simultaneously using multiple oligonucleotides.[41]
5. Conclusion:
The epigenetic pattern is dynamic and gets altered as a response to environment and or several biological processes. Application of epigenetic analysis for forensic investigation has attracted forensic researcher’s attention in the past few decades. Epigenetic applications are new and currently limited in their ability to multiplexing of epigenetic markers. Identification of epigenetic signatures of lifestyle, multiple tissue specific, age-related, disease-associated epigenetic biomarkers, and more advance methods for the extraction of high-quality and quantity of the DNA recovered from crime scene trace are needed for the comprehensive analyses of forensic epigenetics to resolve a crime case and to identify unknown perpetrators of crime.