Success rates of nuclear short tandem repeat typing from different skeletal elements

Aim

To evaluate trends in DNA typing success rates of different skeletal elements from mass graves originating from conflicts that occurred in the former Yugoslavia (Bosnia and Herzegovina and Kosovo) during the 1990s, and to establish correlation between skeletal sample age and success of high throughput short tandem repeat (STR) typing in the large data set of the International Commission on Missing Persons.

Method

DNA extraction and short tandem repeat (STR) typing have been attempted on over 25 000 skeletal samples. The skeletal samples originated from different geographical locations where the conflicts occurred and from different time periods from 1992 to 1999. DNA preservation in these samples was highly variable, but was often significantly degraded and of limited quantity. For the purpose of this study, processed samples were categorized according to skeletal sample type, sample age since death, and success rates tabulated.

Results

Well-defined general trends in success rates of DNA analyses were observed with respect to the type of bone tested and sample age. The highest success rates were observed with samples from dense cortical bone of weight-bearing leg bones (femur 86.9%), whereas long bones of the arms showed significantly lower success (humerus 46.2%, radius 24.5%, ulna 22.8%). Intact teeth also exhibited high success rates (teeth 82.7%). DNA isolation from other skeletal elements differed considerably in success, making bone sample selection an important factor influencing success.

Conclusion

The success of DNA typing is related to the type of skeletal sample. By carefully evaluating skeletal material available for forensic DNA testing with regard to sample age and type of skeletal element available, it is possible to increase the success and efficiency of forensic DNA testing.The aftermath of the 1992-1995 conflict in the area of former Yugoslavia was marked with estimated 40 000 missing individuals. To address the issue of missing persons, the International Commission on Missing Persons (ICMP) was created in 1996 following the G-7 Summit in Lyon, France. ICMP’s mandate was expanded to also cover the DNA typing of missing persons resulting from 1999 conflict in Kosovo region.ICMP employs a “population based, DNA-led” identification system for the identification of missing persons in the region of former Yugoslavia. On a regional scale, DNA profiles from reference samples of living relatives of missing persons are continuously compared in a batch mode to the DNA profiles obtained from mortal remains of victims. To date, more than 84 000 blood samples representing over 28 000 missing individuals have been collected, analyzed, and entered into the database. Since 2001, short tandem repeat (STR) profiles from more than 21 000 skeletal samples, representing more than 15 000 different individuals, have also been entered into the ICMP database (1). DNA matching reports of greater than 99.95% probability of identity have been issued for over 11 600 individuals.ICMP DNA laboratories currently operate at a target rate of 105 bone or tooth extractions per day, using a silica-based extraction method (1-3). Bone and teeth samples tested are between 8 and 15 years post mortem. The quality of DNA preservation in these bones is highly variable and often substantially limited or/and degraded. This reflects the fact that the remains were buried or disposed in many different environmental contexts, with differential exposure to potentially harsh extrinsic factors such as temperature, UV radiation, humidity, and exposure to animals, insects, and microbes. Different disposal conditions are marked by burial in different soil types, complete or partial immersion of remains in water, contact with fire, or use of plastic sheeting. Microbial degradation is variably evidenced in these samples by both bone morphology and co-extraction of sometimes large amounts of microbial DNA (our unpublished observation). As always in this type of work, co-extraction of DNA inhibitors is a serious issue, and is also variable among samples.Bone and teeth samples clearly protect DNA through their physical and/or chemical robustness to environmental degradation and/or biological attack. An elementary manifestation of this is that bone and teeth are often the only surviving material that can be tested. However, the mechanisms by which DNA is preserved in bone are not very well characterized (4). Bone tissue is primarily composed of protein and mineral. The two most abundant proteins in bone tissue are collagen and osteocalcin. Approximately 70% of the mineral portion of the bone is composed of hydroxyapatite, which includes calcium phosphate, calcium carbonate, calcium fluoride, calcium hydroxide, and citrate. Structural arrangement of bone tissue is such that the mineral portion provides structural support to the protein portion in the bone and, by doing so, physically excludes exogenous/extracellular agents/enzymes that are potentially harmful to the protein portion of the bone (4). DNA has a very strong affinity for hydroxyapatite. DNA degradation is linked to the loss of crystallinity in the hydroxyapatite, but it may also be related to the loss of collagen (5).Overall, it seems reasonable to suppose that the characteristics of the bone that are correlated with its general long term survival, ie its resistance to morphological degradation at the macroscopic and microscopic level, would be those that contribute to the protection of DNA from degradation. Bone density, ie the extent of mineralization, is one of the most important intrinsic factors in survival of bone material. There is a significant difference in bone density between men and women, with the latter showing lower density values. The difference in bone density is also specific for different areas of the skeletal element morphology, with the highest density values noted for the mid-shaft region (6). Teeth are the hardest tissue in the human body because of the dental enamel (7).To know which bones best preserve DNA is of fundamental importance to DNA identification casework in mass fatality incidents and mass graves from armed conflict or genocide. The question equally applies to “ancient DNA” analyses in archaeological or human molecular evolutionary investigations. Despite the logical expectation that denser, more intact bones may be preferable, there is very little empirical data published on this issue (8). We also note that a successful recovery of DNA is linked not only to the degree of protection within the bone, but also the total amount of starting DNA. One reason for the lack of precise information on the best samples for DNA testing from degraded bone is the difficulty in performing controlled experiments, with regard to the effect of relevant environmental variables, inter-individual variation (related to for example sex or age), the long periods of time involved, and the need for large sample size.The aim of our study was to analyze DNA typing success rates from very large sample sizes of various skeletal elements from victims of conflict in the former Yugoslavia. Given the large number of variables affecting DNA preservation, a large sample size helps to average out the influences of a wide range of environmental contexts and permit general conclusions. Further, we divided our data into three time periods, with respect to time since death. This allows the analysis of the relative rate of degradation in different skeletal elements over time. These empirical data can serve as a useful guide to sampling strategies from degraded skeletal remains.