Denice Higgins

Teeth as a source of DNA for forensic identification of human remains: A Review

Teeth and bones are frequently the only sources of DNA available for identification of degraded or fragmented human remains. The unique composition of teeth and their location in the jawbone provide additional protection to DNA compared to bones making them a preferred source of DNA in many cases. Despite this, post-mortem changes in the structure and composition of teeth, and the location and diagenesis of DNA within them are poorly understood. This review summarises current knowledge of tooth morphology with respect to DNA content and preservation, and discusses the way in which post-mortem changes will affect the recovery of DNA from teeth under a range of commonly used extraction protocols. We highlight the benefits and pitfalls of using specific tooth tissues for DNA extraction and make recommendations for tooth selection and sampling that will maximise DNA typing success. A comprehensive understanding of tooth structure and an appreciation of the relationship between DNA and mineralized tissues in post-mortem teeth are critical for optimal sample selection. More informed sampling methods that target specific tooth tissues will increase the likelihood of successful genetic analysis and allow for efficient and timely missing persons case work and disaster victim identification response.

Differential nuclear and mitochondrial DNA preservation in post-mortem teeth with implications for forensic and ancient DNA studies.

Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps.

Radiographic Recognition of Dental Implants as an Aid to Identifying the Deceased

Abstract:  This study was undertaken to determine if dental implants can be radiographically differentiated by company type to aid forensic identification of the deceased. Recognition of dental implants on intraoral radiographic images was assessed in a blind study using a radiographic examination guide to highlight differences between dental implants. Inter‐ and intra‐examiner comparisons were conducted and a computer program (Implant Recognition System®) was evaluated to see whether it improved the accuracy of implant recognition. The study found that dental implants could be radiographically differentiated by company type. The Implant Recognition System® in its current form was of little benefit for radiographic assessment of dental implants for forensic odontologists. Prior knowledge of implant types, with a McNemar’s statistical value of 92.9, proved to be most significant in identification.

Targeted sampling of cementum for recovery of nuclear DNA from human teeth and the impact of common decontamination measures

BackgroundTeeth are a valuable source of DNA for identification of fragmented and degraded human remains. While the value of dental pulp as a source of DNA is well established, the quantity and presentation of DNA in the hard dental tissues has not been extensively studied. Without this knowledge common decontamination, sampling and DNA extraction techniques may be suboptimal. Targeted sampling of specific dental tissues could maximise DNA profiling success, while minimising the need for laborious sampling protocols and DNA extraction techniques, thus improving workflows and efficiencies. We aimed to determine the location of cellular DNA in non-degraded human teeth to quantify the yield of nuclear DNA from cementum, the most accessible and easily sampled dental tissue, and to investigate the effect of a common decontamination method, treatment with sodium hypochlorite (bleach).We examined teeth histologically and subsequently quantified the yield of nuclear DNA from the cementum of 66 human third molar teeth. We also explored the effects of bleach (at varying concentrations and exposure times) on nuclear DNA within teeth, using histological and quantitative PCR methods.ResultsHistology confirmed the presence of nucleated cells within pulp and cementum, but not in dentine. Nuclear DNA yields from cementum varied substantially between individuals but all samples gave sufficient DNA (from as little as 20 mg of tissue) to produce full short tandem repeat (STR) profiles. Variation in yield between individuals was not influenced by chronological age or sex of the donor. Bleach treatment with solutions as dilute as 2.5% for as little as 1 min damaged the visible nuclear material and reduced DNA yields from cementum by an order of magnitude.ConclusionsCementum is a valuable, and easily accessible, source of nuclear DNA from teeth, and may be a preferred source where large numbers of individuals need to be sampled quickly (for example, mass disaster victim identification) without the need for specialist equipment or from diseased and degraded teeth, where pulp is absent. Indiscriminant sampling and decontamination protocols applied to the outer surface of teeth can destroy this DNA, reducing the likelihood of successful STR typing results.

Dentine and cementum as sources of nuclear DNA for use in human identification

Teeth are increasingly utilized as a source of nuclear DNA to aid identification of human remains. DNA extraction and the results of genetic analysis from these tissues are extremely variable and to some extent unpredictable. This study examines the availability of nuclear DNA in different areas of the dental hard tissues and explores the extent and nature of the variation within and between individuals. Results of this study indicate that nuclear DNA is available in widely variable quantities in dentine and cementum. This variation exists within teeth and between teeth, even between comparable teeth from the same individual. The quantity of DNA available in dentine is affected by age and dental disease, whereas that in cementum is not. Forensically useful genetic profiles were obtained from as little as 20 mg of tooth powder, thus avoiding the necessity for complete destruction of the tooth. A better understanding of why there is more DNA in one tooth tissue or region compared with another, and of the effects of disease and age, will aid in the selection of which tooth and tissue to sample and will increase the validity of the use of teeth as a source of nuclear DNA for human identification.

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality

Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.

The importance of increasing the forensic relevance of oral health records for improved human identification outcomes

Abstract Dental comparison can confirm human identity to a high degree of certainty. Research examining Australian-made records demonstrated suboptimal recording of dental traits important for forensic dental identification and compliance with Dental Board of Australia (DBA) record keeping guidelines. This is a significant issue for human identification by dental comparison; lack of adequate antemortem information can hinder or obstruct outcomes. Reported identification opinions from the Forensic Odontology Unit of South Australia (FOU-SA) during 2011–2015 were assessed to determine whether the quantitative and qualitative value of antemortem records affected the ultimate identification outcome. Identity was established in 79% (n=197) of the 249 cases presented to the FOU-SA; odontology was unable to categorically confirm an individual’s identity for the remaining 21%. Dental records of almost all cases demonstrated a lack of antemortem data for comparison. Inadequate antemortem information within dental records may preclude identity determination; at minimum, an outcome is hindered by a greater number of issues requiring reconciliation. Given previous results regarding adherence to DBA guidelines, practitioners should reasonably be expected to make small recording changes to improve the continuity of clinical patient care. This antemortem recording improvement will potentially improve the rate at which a forensic identification is reconciled.

A mini-multiplex SNaPshot assay for the triage of degraded human DNA

Short Tandem Repeat (STR) genotyping is currently the primary DNA-based method for human identification, however it can have limited success when applied to degraded human remains. Massively parallel sequencing (MPS) provides new opportunities to obtain genetic data for hundreds of loci in a single assay with higher success from degraded samples. However, due to the extra requirement for specialised equipment, expertise and resources, routine use of MPS may not be feasible or necessary for many forensic cases. Here we describe the development of a mini-multiplex SNaPshot screening tool (Miniplex) for human samples which allows the qualitative comparison of short mitochondrial and nuclear DNA targets, as well as the interrogation of biogeographic ancestry, lineage, and phenotype single nucleotide polymorphisms (SNPs). This tool is useful to triage samples based on sample quality prior to downstream identification workflows and provides broad biological profile data for intelligence purposes.

Development and evaluation of online education to increase the forensic relevance of oral health records

BACKGROUND Human identification can be reliably established by dental comparison; success is significantly impacted by inadequate ante-mortem information. Previous Australian research revealed suboptimal recording of features important for forensic dental identification and compliance with Dental Board of Australia guidelines. We hence created and evaluated an online education programme aimed at improving oral health practitioner recording. METHODS An interactive learning module (ILM) was constructed and released to three focus groups representing practitioners with varying experience levels: Australian Society of Forensic Odontology members, third year dental students and the wider dental community. Pre- and post-participation perceptions were recorded, with percentage, mean, broad agreement, standard deviation and statistical significance between responses determined. RESULTS Improved recognition of importance of record keeping, knowledge, confidence, skill and motivation to learn was seen following ILM interaction. This was particularly significant for students, participants with 3-5 years of experience in their current occupation and those whose highest level of education was achieved in Australia. CONCLUSIONS The ILM increased self-reported awareness, understanding and attitude of participants with different levels of case note recording experience; this can improve recording practises and aid forensic dental identification if utilized in undergraduate teaching and as a continuing professional development tool for dental practitioners.

Stabilisation of dental structures of severely incinerated victims at disaster scenes to facilitate human identification

Fatalities due to fire events such as bushfires, domestic and industrial fires and vehicle accident related incineration, leave victims with limited prospects of being accurately identified. Due to their morphology and anatomical position teeth are uniquely protected in incineration cases and via comparison to dental records often provide the only scientifically valid means of identification. However, extreme heat and direct exposure to flame can render the teeth extremely fragile and vulnerable to damage and loss especially during collection and transportation to the mortuary. Here we highlight the advantages of forensic odontology assistance at the scene of such events and discuss techniques and protocols applied to actual cases in which these processes were used to facilitate the identification of incineration victims.