Kinship analysis of skeletal remains from the Middle Ages

Medieval cemeteries Klisa-Guca Gora, Alihodze and Glavica-Han Bila located in the Travnik area (Travnik, Bosnia and Herzegovina) were archaeologically examined in the period 2011–2014, revealing human skeletal remains of 11 individuals in total. Archaeological skeletal samples, previously deposited in Travnik Homeland Museum (Travnik, Bosnia and Herzegovina) were subjected to genetic analysis. The aim of this research was to test familiar relationship of 11 individuals excavated from three medieval cemeteries and to predict Y-haplogroup for male individuals. In order to perform molecular-genetic characterisation of collected human skeletal remains, two systems of genetic markers were analysed: autosomal and Y-STR loci. Complete or partial data obtained by autosomal STR typing of 11 individuals were subjected to kinship analysis. Male sex was determined in eight samples out of 11. Direct relatives of the "brother-brother" type were detected in one case with high kinship probability (KP) value of 99.99996 %. Complete or nearly complete and usable Y-STR profiles were obtained for six out of eight male individuals. The presence of identical haplotypes at Y-STR loci and results of Y-haplogroup prediction suggest that all male individuals share the same paternal lineage and belong to J2a haplogroup. Overall, this study emphasises the usefulness, efficiency and sensitivity of STR markers in the molecular-genetic characterisation of old skeletal remains as well as the importance of employing additional markers like Y-STRs in archaeogenetic studies, besides traditionally used autosomal STR markers, in order to get a comprehensive information about close and distant relatives, and ancestry.     

The diagnosis of a murder from skeletal remains: a case report

A skeletonised body was found in a Danish forest. The examination of the bones revealed several incisions on the skeleton, one located on the cervical column, two on the sternum, one perforating incision to the right iliac crest, and several superficial ones to the ribs and the right tibia. The skeletonized body was thought to be that of a young man in the twenties with a height of 170 cm. It was estimated that the body had been lying at the spot for at least 1 or 2 y and had been murdered by several stab wounds to the chest and abdomen. Police enquiries subsequently revealed that the deceased was a 23-year-old male with a height of 171 cm. A man confessed to having murdered the victim 11/2 years earlier with several stab wounds to the face/neck, chest, abdominal wall and thigh. Received: 20 February 1996 / Received in revised form: 23 December 1996     

Comparative study of STR loci for typing old skeletal remains with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits

This study highlights a comparative study of short tandem repeat (STR) loci with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits for typing 27 old skeletal remains collected from 100–1000-year-old mass graves in Pakistan. DNA profiles were obtained from minute quantities of DNA (even from?≤?10?pg/μL) with modified protocols of these kits, which is a significant achievement in this study. Consensus profiles were produced for each bone sample. A comparison was carried out between Identifiler and Minifiler successfully genotyped STR loci. Full concordance was perceived in 97.33% (146/150) of the compared STR loci, while discordant STR loci were 2.67% (4/150) of the total successfully genotyped STR loci due to either or both allele drop-out or drop-in. Finally, it was observed that the AmpFlSTR MiniFiler kit promoted the recovery of locus/alleles that failed to type with the AmpFlSTR Identifiler kit and more informative DNA profiles were obtained from old skeletal remains with the AmpFlSTR MiniFiler STR kit compared with the AmpFlSTR Identifiler STR kit.     

Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5′ defined sequence and alter the number of bases on the 3′ end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.     

DNA typing in cases of blood chimerism

A chimera is an organism whose cells derive from two or more distinct zygote lineages. and therefore two different blood cell populations circulate in one individual. To point out the potential pitfalls in forensic analysis, a set of triplets (a girl and two boys) who revealed blood chimerism was investigated with four STR systems using PCR. The results indicated that a DNA profile based on DNA extracted from blood can lead to a false determination because the band pattern of each triplet contained a mixture of the original genotype and the genotype of the siblings. Additional investigations on biological materials other than blood must be made in order to find out the real genetic characteristics of each child. Received: 17 July 1998 / Received in revised form: 27 November 1998     

Autosomal and Y-STR analysis of degraded DNA from the 120-year-old skeletal remains of Ezekiel Harper

The 120-year-old skeletal remains of Confederate Civil War soldier Captain Ezekiel “Zeke” Harper were exhumed by court order in January 2011 for DNA analysis. The goal of the DNA testing was to support or refute whether Captain Harper had fathered a son (Earl J. Maxwell) with his Native American maid prior to his murder in 1892. Bones with adequate structural integrity (left tibia, right tibia, right femur, mandible, four teeth) were retrieved from the burial site and sent to the Institute of Applied Genetics in Fort Worth, Texas for analysis. Given the age and condition of the remains, three different extraction methods were used to maximize the probability of DNA recovery. The majority of the DNA isolates from over fifty separate bone sections yielded partial autosomal STR genotypes and partial Y-STR haplotypes. After comparing the partial results for concordance, consensus profiles were generated for comparison to reference samples from alleged family members. Considering the genetic recombination that occurs in autosomal DNA over the generations within a family, Y-STR analysis was determined to be the most appropriate and informative approach for determining potential kinship. Two of Earl J. Maxwell's grandsons submitted buccal samples for comparison. The Y-STR haplotypes obtained from both of these reference samples were identical to each other and to the alleles in Ezekiel Harper's consensus profile at all 17 loci examined. This Y-STR haplotype was not found in either of two major Y-STR population databases (U.S. Y-STR database and YHRD). The fact that the Y-STR haplotype obtained from Ezekiel's skeletal remains and Earl's grandsons is not found in either population database demonstrates its rarity and further supports a paternal lineage relationship among them. Results of the genetic analyses are consistent with the hypothesis that Earl J. Maxwell is the son of Ezekiel Harper.     

Prediction of autosomal STR typing success in ancient and Second World War bone samples

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5 g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51 ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.     

Development and validation of a new STR 25-plex typing system

In this study, a new STR 25-plex typing system, including 23 autosomal STRs (D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, vWA) and a Y-STR locus of DYS391 and amelogenin, was developed. The included 24 STRs belonged to the main international DNA databases (CODIS, ISSL, ESS-extended, UCL, GCL and NCIDD) except D6S1043 (specially chosen for Chinese population). Developmental validation indicated that the STR 25-plex typing system was reproducible, accurate, sensitive and robust. The sensitivity testing of the system was such that a full profile was obtainable even with 125 pg of human DNA. Specificity testing was demonstrated by the lack of cross-reactivity with a variety of commonly encountered animal species and microbial pool. For the stability testing, full profiles can been obtained with humic acid concentration ≤ 60 ng/μL and hematin < 500 μM. Also, this multiplex system is suitable for mixture study. All of the minor alleles were called for ratios of 1:1, 1:3 and 3:1 of the mixture with the system. In addition, the whole PCR amplification can finish within 1 h, making the system suitable for fast-detection. For the forensic evaluation of the multiplex system, 23 autosomal STRs included followed the Hardy–Weinberg equilibrium. A total of 268 alleles were detected for the 23 autosomal STR loci among 200 individuals. Since 23 autosomal STRs were independent from each other, CMEC_duo was 0.99999916563607 and CMEC_tri was 0.99999999986525. All the forensic efficiency parameters demonstrated that this multiplex system is highly polymorphic and informative in the Han population of China.     

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store.We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA^® cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor^®HY. All samples were amplified using PowerPlex^®21 and PowerPlexY^®23 systems and analyzed using the AB3500 Genetic Analyzer and the GeneMapper^® ID-X v.1.2 software.PCR inhibitors were consistently detected in bone marrow, muscle tissue, rib and vertebra samples. Amplifiable DNA was obtained in a majority of the samples analyzed. DNA recovery from 0.1 g biological material was adequate for successful genotyping of most of the non-bone and bone samples. Complete DNA profiles were generated from bone marrow, femur, metatarsal and patella with 0.1 ng DNA template. Using 0.5 ng DNA template resulted in increased allele recovery and improved intra- and inter-locus peak balance.     

Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system

Retrieving information about externally visible characteristics from DNA can provide investigative leads to find unknown perpetrators, and can also help in disaster victim and other missing person identification cases. Aiming for the application to both types of forensic casework, we previously developed and forensically validated the HIrisPlex test system enabling parallel DNA prediction of eye and hair colour. Although a recent proof-of-principle study demonstrated the general suitability of the HIrisPlex system for successfully analysing DNA from bones and teeth of various storage times and conditions, practical case applications to human remains are scarce. In this study, we applied the HIrisPlex system to 49 DNA samples obtained from bones or teeth of World War II victims excavated at six sites, mostly mass graves, in Slovenia. PCR-based DNA quantification ranged from 4 pg/μl to 313 pg/μl and on an average was 41 pg/μl across all samples. All 49 samples generated complete HIrisPlex profiles with the exception of one MC1R DNA marker (N29insA) missing in 83.7% of the samples. In 44 of the 49 samples (89.8%) complete 15-loci autosomal STR (plus amelogenin) profiles were obtained. Of 5 pairs of skeletal remains for which STR profiling suggested an origin in the same individuals, respectively, 4 showed the same HIrisPlex profiles and predicted eye and hair colours, respectively, while discrepancies in one pair (sample 26 and 43) are likely to be explained by DNA quantity and quality issues observed in sample 43. Sample 43 had the lowest DNA concentration of only 4 pg/μl, producing least reliable STR results and could be misleading in concluding that samples 43 and 26 originate from the same individual. The HIrisPlex-predicted eye and hair colours from two skeletal samples, suggested to derive from two brothers via STR profiling together with a living sister, were confirmed by the living sister's report. Overall, we demonstrate that after more than 70 years, HIrisPlex-based eye and hair colour prediction from skeletal remains is feasible with high success rate. Our results further encourage the use of the HIrisPlex system in missing person/disaster victim identification to aid the identification process in cases where ante-mortem samples or putative relatives are not directly available, and DNA predicted eye and hair colour information provides leads for locating them, allowing STRbased individual identification.     

Anthropological analysis of the Second World War skeletal remains from three karst sinkholes located in southern Croatia

Although in the cases of war crimes the main effort goes to the identification of victims, it is crucial to consider the execution event as a whole. Thus, the goal of the research was to determine the trauma type and probable cause of death on skeletal remains of civilians executed by partisans from WWS found in the three karst sinkholes and to explain the context in which the injuries occurred. We determined biological profiles, pathological conditions, traumas, and assessed their lethality. Nineteen skeletons were found, 68.4% had, at least, one perimortem trauma, classified as lethal/lethal if untreated in 69.2% cases. The type of execution and administered violence showed to be age and health dependent: elderly and diseased were executed with the intention to kill, by the gunshot facing victims, whilst the more violent behavior expressed towards younger and healthy individuals was indicated by the higher frequency of blunt force trauma.     

Isolation and characterization of human DNA from mosquitoes (Culicidae)

Human DNA was prepared from mosquitoes (Culicidae) which were collected in a room shared by four human individuals. Several insects did not contain human blood and DNA preparation from them was not successful. However, high molecular weight human genomic DNA could be isolated from four insects. HLA-DQα and D1S80 analysis showed that the blood from one insect was a mixture from two persons, whereas the others contained blood from single individuals. Human DNA isolated 26 h after ingestion was still suitable for typing. These results showed that DNA isolated from mosquitoes is qualitatively and quantitatively sufficient for DNA typing and could be helpful to identify individuals involved in certain cases of body violence or captivity. Received: 3 November 1997 / Received in revised form: 21 July 1998     

Genetic data obtained for two Chinese Han populations with a quadruplex fluorescent STR typing system (HUMVWA, HUMTH01, D21S11 and HPRT)

DNA typing of four tetrameric repeat loci (HUMVWA, HUMTH01, D21S11 and HPRT) was carried out in a Chinese Han population from Shanghai (East China) and one from Guangzhou (South-East China) using a quadruplex PCR amplification and detection of the fluorescent-labeled alleles on the ALF DNA sequencer. All loci were in accordance with Hardy-Weinberg equilibrium except for D21S11 in the Guangzhou population. A test for population differentiation showed no statistical difference in the allele frequency distribution between the two populations. Comparison of the allele frequency data with other Chinese Han populations from North and South-West China for the STR loci HUMVWA and HUMTH01 revealed heterogeneity between Northern Chinese Han and Southern Chinese Han, which is in accordance with previous studies on the basis of protein markers. Received: 22 December 1997 / Accepted: 23 April 1998     

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs,assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.     

Searching for first-degree familial relationships in California's offender DNA database: validation of a likelihood ratio-based approach

A validation study was performed to measure the effectiveness of using a likelihood ratio-based approach to search for possible first-degree familial relationships (full-sibling and parent–child) by comparing an evidence autosomal short tandem repeat (STR) profile to California's ∼1,000,000-profile State DNA Index System (SDIS) database. Test searches used autosomal STR and Y-STR profiles generated for 100 artificial test families. When the test sample and the first-degree relative in the database were characterized at the 15 Identifiler^® (Applied Biosystems^®, Foster City, CA) STR loci, the search procedure included 96% of the fathers and 72% of the full-siblings. When the relative profile was limited to the 13 Combined DNA Index System (CODIS) core loci, the search procedure included 93% of the fathers and 61% of the full-siblings. These results, combined with those of functional tests using three real families, support the effectiveness of this tool. Based upon these results, the validated approach was implemented as a key, pragmatic and demonstrably practical component of the California Department of Justice's Familial Search Program. An investigative lead created through this process recently led to an arrest in the Los Angeles Grim Sleeper serial murders.     

Developing a DNA methylation assay for human age prediction in blood and bloodstain

Age prediction of an individual based on biological traces remained in a crime scene is of ultimate importance for criminal investigation. Growing evidence indicates that some CpG sites may have age-related methylation changes and thus may be a promising tool for age prediction. In this study, we utilized the pyrosequencing approach to screen age-related CpG (AR-CpG) sites for age prediction. Five AR-CpGs were identified as age-related markers from thirty-eight candidates, among which three CpG sites, ITGA2B_1, NPTX2_3, and NPTX2_4 were never reported in previous studies. We fit a linear regression model for age prediction based on methylation assay for 89 blood samples from donors aged 9–75 years old. The model included four AR-CpG markers in three gene fragments ASPA, ITGA2B and NPTX2 and enabled the age prediction with R² = 0.819. The mean absolute deviation (MAD) from chronological age of the model was 7.870. We validated the linear regression model with a validation set of 40 blood samples, and the prediction MAD was 7.986. There was no statistically significant difference in age prediction between 20 pairs of blood samples and bloodstains. Six pairs of fresh and old bloodstains were analyzed using our assay. The obtained results showed the assay still performed an effective prediction on bloodstains after four-month storage in room conditions. This study indicates that our DNA methylation assay is a reliable and effective method for age prediction for forensic purposes.     

Population data for 15 loci (Identifiler Kit) in a sample from the Valley of Mexico

We analyzed 242 individuals from the Valley of Mexico, including the larger and more cosmopolitan city of this country. They were PCR-typed for 15 STR loci with the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems). Allele frequencies for each STR were estimated and compared to previous reports. Genotype distribution by locus and by two-loci combination was in agreement with Hardy-Weinberg expectations for all fifteen STRs. This STR system in Mexican-mestizos presented a combined probability of exclusion (PE) and discrimination (PD) longer than 99.999%, respectively.     

Sequential multiplex amplification (SMA) of genetic loci: A method for recovering template DNA for subsequent analyses of additional loci

A method called Sequential Multiplex Amplification (SMA) has been developed whereby a limited amount of DNA extracted from a sample can be reutilized for several single polymerase chain reaction (PCR) amplifications. The method involves recovery of genomic template DNA by microfiltration of PCR-amplified samples. Up to 5 different loci have been typed, each in a single system PCR-based assay, beginning with a test quantity of 5 ng template DNA. Genotypes of the DNA donors were compared with those obtained from individual amplifications and shown to be identical. This could be a useful technique for typing a number of loci from a limited amount of DNa and to recover template DNA from samples previously subjected to PCR. Obviously, when small quantities of template DNA are available, this technique can prove quite useful.     

Interpreting a major component from a mixed DNA profile with an unknown number of minor contributors

Modern interpretation strategies typically require an assignment of the number of contributors (N) to a DNA profile. This can prove to be a difficult task, particularly when dealing with higher order mixtures or mixtures where one or more contributors have donated low amounts of DNA. Differences in the assigned N at interpretation can lead to differences in the likelihood ration (LR). If the number of contributors cannot reasonably be assigned, then an interpretation of the profile may not be able to be progressed.In this study, we investigate mixed DNA profiles of varying complexity and interpret them altering the assigned N. We assign LRs for true- and non- contributors and compare the results given different assignments of N over a range of mixture proportions. When a component of a mixture had a proportion of at least 10%, a ratio of at least 1.5:1 to the next highest component, and a DNA amount (as determined by STRmix™) of at least 50 rfu, the LR of the component for a true contributor was not significantly affected by varying N and was therefore suitable for interpretation and the assignment of an LR. LRs produced for minor contributors were found to vary significantly as the assigned N was changed. These heuristics may be used to identify profiles suitable for interpretation.     

Femur, rib, and tooth sample collection for DNA analysis in disaster victim identification (DVI)

Although much literature is available on DNA extraction from tissue samples to obtain the best possible genotyping results, to the best of our knowledge no written recommendations exist on how to excise or extract bone and tooth samples from a victim to facilitate this. Because the possibility of cross-contamination is high, especially when excising numerous samples under disaster conditions, it is important to minimize this risk and to keep samples in optimum condition. In this paper a standard operating procedure is proposed for collection of femur, rib, and tooth samples to aid victim identification both after mass disasters and in (single) forensic investigations.