Research paper
Introducing novel type of human DNA markers for forensic tissue identification: DNA copy number variation allows the detection of blood and semen

https://doi.org/10.1016/j.fsigen.2018.06.021Get rights and content

Highlights

  • Novel type of DNA markers introduced for forensic tissue identification.

  • Copy number variation (CNV) DNA markers identified for blood and semen.

  • CNV qPCR assays show high sensitivity, specificity and coping with degraded DNA.

  • CNV qPCR products represent suitable input material for forensic STR-profiling.

Abstract

Establishing the cellular or tissue-type origin of human biological traces found at crimes scenes is forensically relevant, as it allows evaluating the crime relevance of such traces and enables reconstructing the sequence of crime events. Messenger RNA and micro RNA markers are useful for forensic tissue identification, but provide challenges for linking RNA-identified cell/tissue types with DNA-identified trace donors, especially in mixed traces. DNA methylation markers overcome this problem, but provide technical challenges due to the DNA treatment required by most analysis methods. Here we introduce a novel type of DNA markers for forensic tissue identification analysed without prior DNA treatment, namely copy number variation (CNV). Using genome-wide CNV screening followed-up by targeted qPCR confirmation, and using qPCR analysis of additional CNV-like candidate DNA markers, in samples of several individuals from all commonly encountered forensically-relevant tissue types, we identified DNA markers specific for blood and semen, respectively. Preliminary forensic validation testing demonstrates that the developed qPCR assays are highly sensitive – delivering positive results down to picogram level of input DNA, specific, and can cope well with degraded DNA, providing suitable prerequisites for forensic applications. Moreover, we exemplified that using the CNV qPCR products as input material for subsequent forensic STR analysis delivered full STR profiles, opening-up new avenues of using the same DNA aliquot for both forensic purposes, tissue and individual identification. Provided additional forensic validation studies, we envision the application of these novel DNA markers for forensic tissue identification in future forensic casework. Such CNV markers are particularly useful for tissue identification in old/cold cases, where aged/old DNA extracts are available that contain no RNA and are not suitable for DNA methylation analysis due to limited DNA quantity and quality.

Introduction

Establishing the cellular or tissue origin of human biological traces found at crimes scenes represents a crucial task in forensic casework, because it allows for the reconstruction of the sequence of criminal actions and an evaluation of the relevance of the trace to the crime event. Most human biological materials left at crime scenes include peripheral blood, menstrual blood, semen, saliva, vaginal secretion, skin, and mixtures of any of those. Classical methods used for forensic tissue identification rely on chemical, enzymatic or immunological tests, but most of them are presumptive and prone to false positive or false negative outcomes [1].

Previously developed molecular methods based on messenger (m) RNA or micro (mi) RNA markers, allow for sensitive, specific, non-destructive, and parallel sample analysis [[2], [3], [4]]. RNA-based forensic tissue identification relies on markers in genes with high expression, and thus high mRNA/miRNA copy number, in the target tissue, while low expression/RNA copy number in all non-target tissues that are forensically relevant. However, the high RNA copy number in the target tissue contrasts with the low DNA copy number of STR markers used for forensic DNA profiling i.e., two copies for every diploid cell and one for every haploid cell. This large copy number difference between on one hand m/miRNA molecules used for tissue identification, and on the other hand, DNA molecules used for individual identification, can provide challenges for linking DNA-identified trace donors with RNA-identified tissue types, especially when dealing with mixed traces from various contributors and different tissue types [5]. Therefore, tissue type determination and individual identification should ideally be established from the same molecule, i.e. from DNA.

Recently, DNA methylation markers were proposed for forensic tissue identification based on differences in methylation levels between the targeted and all non-targeted tissues [6], which in principle allow tissue identification and individual identification both being done from DNA. Despite the noticeable progress in the development of this epigenetic approach on forensic tissue identification [7,8], its application remains technically challenging. One problem with most methods currently used for DNA methylation analysis in forensic tissue identification (but likewise any other forensic epigenetic application [9]) is that they rely on prior bisulfite conversion of the DNA, which is of decreased efficiency in decreased amounts of input DNA and additionally leads to DNA degradation [10,11]. Since many crime scene traces contain minute amounts of degraded DNA, all DNA methylation analysis methods that are based on bisulfite conversion are not ideal for forensic applications.

The above-described challenges provided the motivation for the current study to search for alternative DNA markers suitable for forensic tissue identification i.e., DNA markers that are tissue-specific and can be easily quantified with simple, well-established and sensitive methods by avoiding any prior DNA treatment. Despite the general belief, DNA content in different cells of the same individual not always is the same for all parts of the DNA, a phenomenon known as somatic mosaicism [12]. Most of somatic DNA mosaicisms are due to random mutations accumulating during cell divisions, with one of these mutation classes being copy number variations (CNVs). In principle, CNVs with strong differences between forensically relevant tissue types represent potential DNA marker resources for forensic tissue identification. However, CNVs were not explored in the context of forensic tissue identification as of yet. With the current study, we present the first empirical evidence demonstrating that CNVs analysed by qPCR represents a suitable and promising DNA approach for forensic tissue identification, which allows tissue and individual identification to be obtained from the same nucleic acid.

Section snippets

Sample collection and DNA isolation

Peripheral blood (n = 125), saliva (n = 34), semen (n = 23), menstrual blood (n = 26), vaginal secretions (n = 24), and palmar skin (n = 5) samples were collected from healthy volunteers of European origin under informed consent (Table 1). Peripheral blood was collected by venipuncture in BD Vacutainer® tubes, or with finger pricks, and subsequently absorbed by cotton swabs. For saliva collection, volunteers were asked to avoid eating and drinking for at least half an hour before sampling.

Array CGH analysis in forensically-relevant tissues to identify tissue-specific CNVs

By use of array CGH in DNA samples from different forensically-relevant tissue types i.e., peripheral blood, menstrual blood, saliva, vaginal secretion and semen, of seven individuals (4 females and 3 males) to search for tissue-specific CNVs, two major regions of genomic copy number alterations were found for all hybridizations of blood (peripheral and menstrual) against all other tissues tested. These two regions were located in chromosomes 2 and 14, and approximately correspond to 2p11.2 and

Conclusions

Our study highlights a novel type of DNA markers suitable for forensic tissue identification by avoiding the problem of bisulfite conversion as seen with DNA methylation markers. We provide the first evidence that blood and semen origin of human biological traces, such as those found at crime scenes, can be established from DNA with CNV or CNV-like markers using simple, sensitive and reliable qPCR technology. We demonstrated that cjKREC and cjTREC CNV markers can reliably identify blood and can

Acknowledgments

We are grateful to all volunteers for providing samples. MK, DZ and IK were supported by Erasmus MC. MK and DZ were additionally supported by the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the frameworks of the Forensic Genomics Consortium Netherlands (FGCN). JCC was supported by the European Social Fund within the framework of the Human Capital Operational Programme.

References (34)

  • D. Zubakov et al.

    MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

    Int. J. Leg. Med.

    (2010)
  • S. Forat et al.

    Methylation markers for the identification of body fluids and tissues from forensic trace evidence

    PLoS One

    (2016)
  • A. Vidaki et al.

    From forensic epigenetics to forensic epigenomics: broadening DNA investigative intelligence

    Genome Biol.

    (2017)
  • K. Munson et al.

    Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR

    Nucleic Acids Res.

    (2007)
  • A. Piotrowski et al.

    Somatic mosaicism for copy number variation in differentiated human tissues

    Hum. Mutat.

    (2008)
  • R. Pique-Regi et al.

    Sparse representation and Bayesian detection of genome copy number alterations from microarray data

    Bioinformatics

    (2008)
  • D.C. Douek et al.

    Changes in thymic function with age and during the treatment of HIV infection

    Nature

    (1998)
  • Cited by (13)

    • Unlocking the potential of forensic traces: Analytical approaches to generate investigative leads

      2022, Science and Justice
      Citation Excerpt :

      The bisulfite conversion is troublesome since it requires large amounts of DNA, something that is not always feasible in forensic investigation, where the amount of sample available is limited [77,78]. Because of this, alternative methods have also been evaluated, such as Copy Number Variation (CNV) analysis, based on DNA varying between cell types due to somatic mosaicism [79]. Other researchers during the late 2010s have stepped away from human DNA and rather focused onto body fluid identification through the taxonomical profiling of bacterial populations.

    • Effect of indoor environmental exposure on seminal microbiota and its application in body fluid identification

      2020, Forensic Science International
      Citation Excerpt :

      Since that, forensic scientists are exploring more new biology technology to identify semen stains. A novel type of mtDNA copy number and telomere repeat length were used to identify semen, but it limit on immature sperm cells and azoospermic samples [15]. Furthermore, a novel method based on MALDI-MS was available for distinguish human body fluid rapidly [16].

    • Evaluation of a co-extraction kit for mRNA, miRNA and DNA methylation-based body fluid identification

      2020, Legal Medicine
      Citation Excerpt :

      Moreover, considering the quality and quantity of eluted DNA and the volumes used in the DNAm analysis, DNA typing for human identification can be performed using the same DNA eluates. In addition to mRNA, miRNA and DNAm, other molecules, such as bacterial DNA [45–48], circular RNA [49,50] and DNA copy number variation [51], have been recently reported as novel BFID markers. Although further modification may be necessary, the APU may also be used for these BFID methods.

    • Expression profile analysis of piwi-interacting RNA in forensically relevant biological fluids

      2019, Forensic Science International: Genetics
      Citation Excerpt :

      Conventional methods for body fluid identification were majorly based on enzymatic or immunological tests, which are comparatively limited in sensitivity and specificity [2]. Over the last decade, some new approaches, including DNA-methylation pattern and copy number variation analysis, microbial signature assay and RNA profiling analysis, have been proposed as alternative methods in discriminating body fluids [3–5]. For the most common forensically body fluids/tissues (venous blood, menstrual blood, saliva, semen, vaginal secretion and skin), several message RNA (mRNA) marker combinations have been evaluated by the European DNA profiling group (EDNAP) laboratories with capillary electrophoresis (CE)-based analysis or massively parallel sequencing (MPS) [6–10].

    View all citing articles on Scopus
    View full text