Exploring the Value of Biogeographic Ancestry in the Absence of a DNA Database

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.

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.

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.