Forensic Genetic Investigation of Unidentified Human Skeletal Remains Discovered in the Himalayas:
Exploring the Value of Biogeographic Ancestry in the Absence of a DNA Database
Dr. Angie Ambers, University of New Haven
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There are many scenarios encountered in forensic casework in which bone may be the only viable sample type for DNA testing, including fires, terrorist attacks, natural disasters, war conflicts, airline crashes, homicides, human trafficking, and mass graves. In these cases, the condition of recovered human remains can range from relatively intact to highly degraded and/or fragmented. In some homicides, perpetrators dismember the bodies of their victims and hide or disperse the individual parts across a wide geographic region in order to impede discovery of the remains, as well as to hinder subsequent identification efforts. Additionally, in terms of forensic investigation, unidentified remains in an advanced state of decomposition often essentially become ‘skeletal’ cases because the soft tissue is too decomposed to obtain usable data for positive identification. Decomposition of soft tissue prevents visualization of distinguishing tattoos or scars on the skin, and can nullify the possibility of a fingerprint match or facial feature recognition. In these cases, bone is the most viable sample for DNA testing and therefore may be the only avenue for identifying a decedent.
In the U.S., thousands of children and adults vanish without a trace and/or under suspicious circumstances every year. The latest statistics available indicate that the National Crime Information Center (NCIC) contains 88,089 active missing persons records and a registry of 8,634 unidentified (skeletonized) remains. However, the actual number of unidentified skeletal remains that exists likely is substantially higher, given that many victims remain undiscovered to date.
Fortunately, the U.S. has a well-established database containing DNA profiles of missing persons and/or their relatives, for comparison when skeletal remains are recovered and to assist in identification efforts. However, although forensic DNA typing is widely conducted around the world, many countries still have not developed and/or populated reference DNA databases to assist in cases involving unidentified human remains (UHR).
India “missing persons” case: Skeletal remains discovered in the Dhauladhar Range of the western Himalayas (Himachal Pradesh)
In 2017, I traveled twice to the Gurgaon/Gurugram region of Delhi, India to train scientists from various Indian states and the Maldives Police Service on the processing of bone samples in forensic DNA casework. During the workshop training, I had the opportunity to work on an unidentified skeletal remains case with Dr. Vivek Sahajpal and Dr. Arun Sharma, two exceptional forensic DNA scientists from the State Forensic Science Laboratory in Himachal Pradesh.
Himachal Pradesh is a northern state of India, situated in the western Himalayas. Most of the state lies in the foothills of the Dhauladhar Range, with a scenic landscape of snow-clad mountains, deep gorges, densely forested valleys, large lakes, and cascading streams (Figure 1). Due to the majesty of the region, tourism is a major contributor to the state's economy and growth. Popularly referred to as “The Land of Wanderlust,” it is a well-known trekking and climbing destination for adventure- and spiritual seekers. Additionally, Himachal Pradesh is the home of the Dalai Lama and the Tibetan government-in-exile. The strong Tibetan presence is reflected in the state’s Buddhist temples and monasteries, as well as during its vibrant festivals and celebrations.
In 2016, human skeletal remains were discovered along Indrahar Pass, a mountain pass in the Dhauladhar range of the Himalayas, located at an altitude of 4,342 meters (14,245 ft) above sea level. Indrahar Pass is near the town of Dharamshala in Himachal Pradesh and attracts substantial tourist traffic during the trekking season (April – October). In the last two decades, approximately 30 foreign tourists have gone missing in Himachal Pradesh, notably near the tourist hotspot of Dharamshala. The majority of these tourists were of European descent, including trekkers from the United States, Australia, France, England, Russia, Italy, Sweden, Ireland, Yugoslavia, and the Netherlands. When unknown skeletal remains were discovered in 2016, Indian authorities understandably assumed that the remains belonged to one of these missing tourists, and implored forensic scientists to attempt to recover DNA in an effort to identify the decedent.
Figure 1. Map showing the location of Himachal Pradesh, a northern state of India situated in the middle of the western Himalayas. This scenic “land of wanderlust” is a popular tourist destination for adventure seekers and trekkers. In 2016, human skeletal remains were discovered along Indrahar Pass in the Dhauladhar range of the Himalayas, near the town of Dharamshala.
The skeleton discovered was incomplete, consisting only of disarticulated cranial plates, a lower mandible (sans teeth), two femora, one tibia, the diaphyses of five unassigned long bones, and severely degraded fragments of a pelvis. Additionally, four disarticulated (loose) molar teeth were recovered from the scene.
The gender of the remains was unknown prior to DNA testing. Anthropological gender assessment can be challenging without access to the cranium and the pelvis, two of the most sexually dimorphic skeletal elements in the human body. In this case, the skull and pelvis were not intact, and only portions of each were recovered. Cranial plates were disarticulated and fragmented, and only portions of the highly degraded pelvis were found. Therefore, it was necessary to determine the gender of the decedent using genetic analyses.
In addition to the poor condition of the skull and pelvis, many of the long bone epiphyses were missing or severely degraded, likely from advanced bone diagenesis due to environmental exposure. Bryophyta (moss) was growing on the epiphyses of a few of the long bones recovered. Two different long bones were selected for DNA testing after extensive visual examination to determine the most viable candidate samples for typing success. These two bones were the most structurally intact, with no visible fractures to the diaphysis, and appeared to contain the most dense cortical bone material, as estimated by comparative manual handling of all available bones in the sample set. Another (larger) long bone in the sample set was not selected for testing due to a significant longitudinal fracture that extended along the length of the majority of the diaphysis, which likely would have compromised the quality of DNA within the bone microstructure over time.
Multiple DNA extractions using 0.5g−1.0g bone or tooth powder were performed in a designated low copy number (LCN) area of the Thermo Fisher Scientific laboratory in the Gurgaon/Gurugram region of Delhi, India. DNA quantification and autosomal STR genotyping were conducted using the Quantifiler® Trio and GlobalFiler™ kits (Thermo Fisher Scientific, Waltham, MA), respectively. In total, extractions were performed on nine different bone cuttings and two molar teeth. A complete 24-locus consensus profile was obtained, with complete concordance in allele calls between the various bone sections and teeth tested. Moreover, DNA was not detected in any of the extraction reagent blanks or amplification negative controls. A female analyst (myself) conducted all testing for each set of remains, including surface cleaning, bone grinding, DNA extractions, quantification, PCR amplifications, and genotyping. Results confirmed that the unknown skeletal remains were male.
Although the decedent’s endogenous STR profile had been determined, a DNA database was not available for kinship analysis and/or comparison to exemplars. At this stage, the only feasible option for identifying this individual involved reaching out to government officials in the “home” countries of each of the 30+ missing tourists and requesting that family reference samples be collected for comparison to the profile obtained from the skeletal remains. Aside from logistical challenges in undertaking this extensive effort, there are sensitivity issues that must be considered with such an approach. The families of these 30+ missing persons had already experienced a tremendous amount of trauma and grief due to unanswered questions associated with the disappearance of their loved one. Although discovery of a loved one’s remains and the ability to provide a proper burial often can provide some measure of “closure,” the reality in this case is that we had no reasonable level of confidence in assuming that the remains belonged to one of these families.
Assessment of biogeographic ancestry: Y-STRs and ancestry-informative SNPs (aiSNPs)
Since STR genotyping revealed that the remains were male – and Y haplogroups can be used to infer biogeographic ancestry – a decision was made to perform additional testing with markers that are specific to the Y chromosome (using the 27-locus Yfiler™ Plus multiplex by Thermo Fisher Scientific). With the alleles in the decedent’s Y-STR haplotype, Y haplogroup prediction was performed using the ancestry feature and metapopulation tool of the Y-STR Haplotype Reference Database (YHRD). The YHRD-based prediction was cross-checked with HaploGrep software. Analysis of the Y-STR alleles in the profile obtained from the skeletal remains revealed that the decedent was of Asian descent (not European), as shown in the heat map and diagram generated by YHRD (Figure 2).
Figure 2. Biogeographic ancestry prediction for unidentified skeletal remains discovered in the western Himalayas of India, using the ancestry feature and metapopulation tool of the Y-STR Haplotype Reference Database (Y-HRD) (accessed 10-18-2017). The remains were originally presumed to be of one of the 30+ European and American tourists who have gone missing in the last two decades while trekking in Himachal Pradesh.
In addition to traditional capillary electrophoresis (CE)-based methods for Y-chromosome typing and the utility of Y-STR markers for inferring biogeographic ancestry, the advent of massively parallel sequencing (MPS) technology has greatly expanded the tools available to scientists for predicting the ancestral origin of unidentified skeletal remains. In this case, samples from the decedent were further tested using the ForenSeq™ DNA Signature Prep Kit (Verogen, San Diego, California USA) and the MiSeq™ MPS platform. The ForenSeq™ panel co-amplifies 24 Y-STRs, 95 human identity SNPs, 56 ancestry-informative SNPs, 22 phenotype-informative SNPs, 29 global autosomal STRs (plus amelogenin), and 7 X-STRs.
The ForenSeq™ Y-STR data were concordant with the Y-STR haplotype obtained using the traditional CE-based method. However, the additional ancestry-informative SNP (aiSNP) data provided an opportunity to support or refute the ancestry prediction previously inferred using YRHD. Of the 56 aiSNPs in the ForenSeq™ panel, results were obtained for 43 loci and were analyzed using the Forensic Research/Reference on Genetics-knowledge base (FROG-kb). FROG-kb (introduced in 2012) is an open access web tool that utilizes SNP data to infer biogeographic ancestry of an unknown sample via comparison to known population data sets contained within the system. The aiSNPs obtained from the skeletal remains discovered in Himachal Pradesh were compared to 139 reference populations in FROG-kb. Statistical calculations support that the decedent is of Asian descent, which is consistent with the ancestry prediction by YHRD. Figure 3 shows the top 10 most likely populations of origin for the unknown decedent, along with statistical likelihoods.
Figure 3. Biogeographic ancestry prediction for unidentified human skeletal remains discovered in the western Himalayas of India, via comparison of endogenous ancestry-informative SNP (aiSNP) data to 139 reference populations in the Forensic Research/Reference on Genetics-knowledge base (FROG-kb) (http://frog.med.yale.edu/). The top 10 FROG-kb results are shown, indicating the most likely populations of origin for the remains.
Ultimately, although the identity of this individual remains unknown to date, the possibility of causing further emotional trauma to families of 30+ missing European and American tourists was circumvented due in large part to recent advances in forensic DNA technology. Although massively parallel sequencing (MPS) is also known as next-generation sequencing (NGS), the reality is that it is no longer “next generation.” Research laboratories have been using the technology for years, and many of the accompanying assays/kits have been through the validation process in preparation for implementation into forensic DNA casework. The changing face of forensic genetics now offers the capability to obtain biogeographic ancestry and phenotype information from unknown samples, which could provide important investigative leads in cases that otherwise may have progressed in the wrong direction. The capability to obtain far more information from an evidentiary sample than was previously possible in the traditional casework landscape likely will alter the way forensic genetic investigations are approached in the future, particularly with unidentified human remains (UHR).
DNA testing of these unidentified remains discovered in the Himalayas was supported by Thermo Fisher Scientific, the State Forensic Science Laboratory (Himachal Pradesh, India), and the UNT Center for Human Identification. I am grateful to Dr. Sahajpal and Dr. Sharma for entrusting me with this case. It was an honor to work with such talented scientists, and to acquire new friends in the process. Lastly, many thanks to Len Goren, Yogeendra Dawalkar, Atima Agarwal, and Jeet Singh at the Thermo Fisher Scientific laboratory in India for their kindness, technical assistance, and support.
Dr. Ambers holds a Ph.D. in molecular biology (with emphasis in forensic genetics and human identification), as well as master’s degrees both in forensic genetics and in criminology. She is currently an Associate Professor in the Forensic Science Department of the Henry C. Lee College of Criminal Justice and Forensic Sciences, at the University of New Haven. She began her career in forensic genetics studying under the late Arthur Eisenberg, one of the pioneers of forensic DNA testing in the United States.
Dr. Ambers worked as a full-time forensic geneticist for the University of North Texas (UNT) Center for Human Identification for 8 years, specializing in DNA analysis and genetic characterization of unidentified human remains (UHR). In 2018, she received the “Outstanding Forensic Genetics Alumni” award for her successes and contributions to the field. Her research has involved an investigation of alternative methods (e.g., whole genome amplification, DNA repair) for improving autosomal and Y-STR typing of degraded and low copy number (LCN) DNA from human skeletal remains and environmentally-damaged biological materials, as well as development/optimization of a DNA-based multiplex screening tool for the separation of fragmented and commingled skeletal remains in mass graves.
Prior to joining the Henry Lee College, Dr. Ambers was an adjunct professor at the University of North Texas for 12 years (teaching molecular biology, genetics, heredity, human anatomy/physiology). In 2008, she developed the curriculum for a course in forensic molecular biology, and for a decade taught DNA analysis/methodology to undergraduate students enrolled in UNT’s FEPAC-accredited forensic science certificate program.
Before pursuing her doctorate, Dr. Ambers was lead DNA analyst and lab manager of the UNT DNA Sequencing Core Facility. Her casework has involved DNA testing of an American Civil War guerrilla scout, several Finnish World War II soldiers, unidentified late-19th century skeletal remains discovered by a construction crew in Deadwood, South Dakota, unidentified skeletal remains of Special Operations soldiers killed during the 1974 Turkish invasion of Cyprus, skeletal remains of two soldiers from Napoleon’s army, skeletal remains exhumed from Prague Castle in the Czech Republic, skeletal remains of soldiers from the Seven Years’ War (1756-1763), and bone samples purported to belong to a member of Jesse James gang (killed during a bank robbery in 1876).
Dr. Ambers co-presented a workshop on “Advanced Methods for DNA-based Analysis of Skeletal Remains” at the 26th International Symposium on Human Identification, and in 2015 was an invited speaker at an international bone workshop/conference in Prague, Czech Republic. In 2017, she traveled twice to India to train scientists from various Indian states and the Maldives Police Service on the processing of bone samples in forensic DNA casework. She specializes in characterization and identification of contemporary, historical, and archaeological human skeletal remains. Her casework and research has been published in various peer-reviewed journals [Forensic Science International: Genetics (FSI: Genetics), International Journal of Legal Medicine, Legal Medicine, BMC Genomics, Croatian Medical Journal, The Journal of Heredity] and has received press in numerous local and national newspapers, including The Washington Times. Dr. Ambers’ most recent casework includes DNA analyses of five sets of human skeletal remains associated with the French explorer La Salle’s last expedition, and twenty-five sets of skeletal remains associated with Spanish royalty and the Kings of Aragon.
In addition to skeletal remains cases and research, Dr. Ambers collaborated with the National Institute of Justice (NIJ) and the Forensic Technology Center of Excellence (FTCoE) in 2014 to develop and disseminate a formal report on the use of “Familial DNA Searching” in cold case investigations, an approach which recently garnered media attention for its use in catching the Golden State Killer. She also recently (2017-2018) served as project lead on a U.S. State Department grant to combat human trafficking in Central America through the application of forensics. As part of the program objectives, she traveled to three Northern Triangle countries (Guatemala, El Salvador, Honduras) to perform gap assessments of government laboratories and train personnel in forensic DNA analysis, with the goal of promoting quality casework methods based on ISO 17025 standards. In addition to providing both lecture and tactile training on DNA analysis methods to Northern Triangle laboratories, Dr. Ambers was part of a consortium to help these countries develop and maintain forensic DNA databases to assist in the identification of missing persons related to human trafficking.