DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of ?eri?i, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex^® Fusion and PowerPlex^®Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains.     

Forensic Identification of Indian Snakeroot (Rauvolfia serpentina Benth. ex Kurz) Using DNA Barcoding

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species‐specific DNA polymorphisms. We found two species‐specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species‐specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot.     

Forensic Use of the Piracatinga Fish (Callophysius macropterus) to Locate and Identify Human Remains Retrieved From the Amazon River

Piracatinga (Callophysius macropterus) are a type of bottom feeder catfish encountered in the Amazon River and its tributaries. We report two cases in which human remains were first located based on a characteristic circular distortion of the surface of the river that the Piracatinga make while they feed. Human skin samples of one of the victims recovered from the Piracatinga digestive tract were subjected to mitochondrial DNA analysis that allowed identification of the body of Case 1; the family recognized body parts of Case 2. Importantly, the location of human body parts and their identification based on DNA analysis enabled the respective families to obtain a death certificate expeditiously in the absence of identifiable remains—a process that normally requires 5 years under current Brazilian law, and in the absence of closure, imposes severe emotional stress on the family of the deceased.     

Integrated DNA and Fingerprint Analyses in the Identification of 60‐Year‐Old Mummified Human Remains Discovered in an Alaskan Glacier

Abstract: This report describes the identification of a merchant mariner who perished in 1948 when Northwest Airlines Flight 4422, a DC‐4 carrying 24 seamen and six crew members crashed into Mount Sanford, Alaska. Fifty‐one years later, a human forearm and hand were found close by the wreckage of the plane, prompting identification efforts using DNA and fingerprints. There were significant challenges to both the fingerprint and DNA analyses. The hand was badly desiccated, making fingerprint friction‐ridge detail almost invisible and the remains had been embalmed upon discovery, making DNA amplification difficult. We present the results of an interdisciplinary approach that successfully addressed these challenges and ultimately led to the identification of the remains. These efforts relied on efficient fingerprint rejuvenation and imaging techniques that improved print resolution, as well as new DNA extraction techniques optimized for aggressively embalmed remains.     

Positive Personal Identification of Human Remains Based on Thoracic VertebralMargin Morphology

Abstract: Radiography has long been used by anthropologists to establish positive personal identification of human remains in forensic cases. These methods have been largely ad hoc and depend upon specific congenital or pathological bone markers. Court rulings, such as Daubert and Mohan have, however, pushed the discipline toward more statistically supportable methods of identification. This study describes the use of normal morphological variation of the thoracic vertebrae to identify human remains. Radiographs from healthy, male individuals, aged 18–55 were examined to identify normally varying features of vertebral morphology. The frequency of occurrence of these features was calculated, tested, and found to be stable in the given sample. The frequencies were compared to establish which sets of traits varied independently of one another. Finally, unknown radiographs were compared to known samples to test the applicability of this method in determining positive identification, with 21 of 24 (87.5%) unknown radiographs positively identified.     

Forensic or Archaeological Issue: Is Chemical Analysis of Dental Restorations Helpful in Assessing Time Since Death and Identification of Skeletonized Human Remains?

In 2011, small mass grave with completely skeletonized remains was discovered in Belgrade suburb. An eyewitness claimed that skeletons belonged to German soldiers killed in WWII. Anthropologists were engaged to investigate whether the skeletal remains correspond to the indicated German group or represent more recent case requiring court trial. Numerous dental restorations were noticed. Owing to the fact that different dental materials were used in dental practice at certain times, the aim of this study was to explore whether analysis of dental restorations could help in identification and estimation of time since death. Inductively coupled plasma optical emission spectrometry revealed that dental fillings corresponded to copper amalgam, conventional silver amalgam, silicophosphate cement, and zinc phosphate cement. Chemical results combined with anthropological and historical facts suggest that the individuals lived before the 1960s in country with well‐developed dental service at that time. Therefore, chemical analysis of dental fillings was useful to distinguish between skeletal remains that are too old to be of forensic interest and the remains relevant to legal investigations.     

Implant Bone Integration Importance in Forensic Identification

Odontological identification consists of the comparison of antemortem dental information regarding a missing person with postmortem data from an unidentified corpse or human remains. Usually, the comparison concerns morphologic features that the operator chooses among all the visible characteristics because of inter‐individual uniqueness; for this reason, implants can be of enormous assistance. A case concerning the recovery of a burnt oral implant, connected to a bone fragment, among 2780 charred bone fragments, suspected to have belonged to a victim of homicide, is presented to demonstrate that dental implants and their site of bone integration represent a very precious element for personal forensic identification. Because of their morphological invariability in time and because of their morphologic uniqueness, they were used as evidence to associate unidentified human charred remains to a missing person where DNA analysis failed to do so. The case illustrates the fundamental contribution, not yet described in literature, given by the clinical aspects of tooth replacement with dental implants to a forensic discipline. Clinical practitioners should therefore be aware of the great importance of their work and of dental records in a forensic identification scenario.     

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*†

Abstract: Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C‐stretch and CA repeat. To observe which CA “alleles” were present in each tissue, PCR products were cloned and re‐sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.     

Morphometric Comparison of Clavicle Outlines from 3D Bone Scans and 2D Chest Radiographs: A Shortlisting Tool to Assist Radiographic Identification of Human Skeletons

This paper describes a computerized clavicle identification system primarily designed to resolve the identities of unaccounted‐for U.S. soldiers who fought in the Korean War. Elliptical Fourier analysis is used to quantify the clavicle outline shape from skeletons and postero‐anterior antemortem chest radiographs to rank individuals in terms of metric distance. Similar to leading fingerprint identification systems, shortlists of the top matching candidates are extracted for subsequent human visual assessment. Two independent tests of the computerized system using 17 field‐recovered skeletons and 409 chest radiographs demonstrate that true‐positive matches are captured within the top 5% of the sample 75% of the time. These results are outstanding given the eroded state of some field‐recovered skeletons and the faintness of the 1950's photofluorographs. These methods enhance the capability to resolve several hundred cold cases for which little circumstantial information exists and current DNA and dental record technologies cannot be applied.     

Genetic Identification of Communist Crimes’ Victims (1944–1956) Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military Cemetery in Warsaw,Poland

As the result of the communist terror in Poland, during years 1944–1956 more than 50,000 people died. Their bodies were buried secretly, and most places are still unknown. The research presents the results of identification of people buried in one of many mass graves, which were found at the cemetery Pow?zki Military in Warsaw, Poland. Exhumation revealed the remains of eight people, among which seven were identified genetically. Well‐preserved molars were used for the study. Reference material was collected from the closest living relatives. In one case, an exhumation of victim's parents had to be performed. DNA from swabs was extracted with a PrepFiler^® BTA Forensic DNA Extraction Kit and organic method. Autosomal, Y‐STR amplification, and mtDNA sequencing were performed. The biostatistical calculations resulted in LR values from 1608 to 928 × 10¹⁸. So far, remains of more than 50 victims were identified.     

Characterization of Blue Pigments Used in Automotive Paints by Raman Spectroscopy

Micro‐Raman spectroscopy was applied to forensic identification of pigments in paint chips and provided differentiation between paint samples. Sixty‐six blue automotive paint samples, 26 solid and 40 metallic were examined. It was found that the majority of the collected Raman spectra provided information about the pigments present. However, in some cases, fluorescence precluded pigment identification. Using laser excitation at longer wavelengths or pretreatment to effect photobleaching often resulted in reduced fluorescence, particularly for solid color samples, and allowed pigment identification. The examined samples were compared pairwise taking into account number, location, and intensity of absorption bands in their infrared spectra. The estimated discrimination power ranged from 97% for solid paint samples to 99% for metallic paint samples.     

The Effects of Different Maceration Techniques on Nuclear DNA Amplification Using Human Bone

Abstract: Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow‐up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpF?STR^® COfiler^® and AmpF?STR^® Profiler Plus^® ID kits. Results showed that heat treatments via microwave or Biz/Na₂CO₃ in sub‐boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long‐term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

DNA Preservation in Skeletal Elements from the World Trade Center Disaster: Recommendations for Mass Fatality Management*,†

Abstract: The World Trade Center (WTC) victim identification effort highlights taphonomic influences on the degradation of DNA from victims of mass fatality incidents. This study uses a subset of the WTC‐Human Remains Database to evaluate differential preservation of DNA by skeletal element. Recovery location, sex, and victim type (civilian, firefighter, or plane passenger) do not appear to influence DNA preservation. Results indicate that more intact elements, as well as elements encased in soft tissue, produced slightly higher identification rates than more fragmented remains. DNA identification rates by element type conform to previous findings, with higher rates generally found in denser, weight‐bearing bones. However, smaller bones including patellae, metatarsals, and foot phalanges yielded rates comparable to both femora and tibiae. These elements can be easily sampled with a disposable scalpel, and thus reduce potential DNA contamination. These findings have implications for DNA sampling guidelines in future mass fatality incidents.     

DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala

No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.     

Intra‐ and Inter‐Element Variability in Mitochondrial and Nuclear DNA from Fresh and Environmentally Exposed Skeletal Remains,

Successful identification of skeletonized remains often relies upon DNA analyses, frequently focusing on the mid‐diaphysis of weight‐bearing long bones. This study explored intra‐bone DNA variability using bovine and porcine femora, along with calcanei and tali. DNA from fresh and short‐term environmentally exposed bone was extracted utilizing demineralization and standard lysis buffer protocols, and DNA quantity and quality were measured. Overall, femoral epiphyses, metaphyses, and the tarsals had more nuclear and mitochondrial DNA than did the femoral diaphyses. DNA loss was much more rapid in buried bones than in surface exposed bones, while DNA quality differed based on environment, but not bone region/element. The demineralization protocol generated more DNA in some bone regions, while the standard lysis was more effective in others, and neither significantly affected DNA quality. Taken together, these findings reinforce the importance of considering inter‐ and intra‐bone heterogeneity when sampling skeletal material for forensic DNA‐based identifications.     

Investigation of Different Ways in Which the CODIS 7.0 May be Used in Mass Disaster Identification

DNA analysis is a key method for the identification of human remains in mass disasters. Reference samples from relatives may be used to identify missing persons by kinship analysis. Different methods of applying the CODIS in disaster victim identification (DVI) were investigated. Two searches were evaluated: (i) relating family relatives to a pedigree tree (FPT) and (ii) relating unidentified human remains to a pedigree tree (UPT). A joint pedigree likelihood ratio (JPLR) and rank were calculated for each search. Both searches were similar in average JPLR and rank. In exceptional cases, namely the existence of a mutation different from the CODIS model, a nonbiological father, a mistake in STR, or incorrect profile association, the UPT search returned one true rank, whereas the FPT search returned no results. This paper suggests a novel strategy to overcome these limitations and increase efficiency in conducting identification of mass disaster victims.     

A Grass Molecular Identification System for Forensic Botany: A Critical Evaluation of the Strengths and Limitations*

Abstract: Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA‐based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single‐nucleotide polymorphisms were utilized as molecular markers for subfamily to species‐level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated “proof of concept” of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.