Abstract
During the military governments in Argentina in the 1970s, the bodies of people who had been detained and later killed were deposited in individual graves as well as common graves. The identity of the majority of the bodies was not provided.
During 2008, the Argentine Forensic Anthropology Team (EAAF) processed nine bone fragments from bodies found in a group of graves in the Province of Buenos Aires.
Nuclear DNA (nDNA) profiles were obtained by amplifying autosomal STRs (using the Identifiler®, Profiler® and COfiler® kits), Y-STR (Y-Filer®) and mini-STR using kits developed “in-house”. The mitochondrial DNA (mtDNA) HV1 and HV2 regions were also sequenced on the same bone samples.
Blood samples were analyzed from 21 individuals who reported to the EAAF that they had a family member missing from the aforementioned time period.
This study presents the results of the comparison made between the bone sample genetic profiles and those of the family members, based on the genetic marker used (STR, Y-STR and/or mtDNA) and on the proposed kinships for each hypothesis. From the 9 bone samples analyzed, a total of 8 individual skeletons were observed. Of those 8 individuals, 4 were identified through kinship analysis.
Keywords
1. Introduction and objectives
Unidentified human remain inhumation in Argentina during the 1970s presented the challenge of identifying those remains through anthropological and DNA studies.
During the year 2008, the investigation of 8 individual graves found by the Argentine Forensic Anthropology Team (EAAF) in a cemetery in the Province of Buenos Aires was started. The 30-year-old skeletons that were recovered from the graves presented perimortem injuries made by firearm projectile. Bone remains were poorly preserved due to the characteristics of the soil.
Anthropological studies allowed for the individualization of eight skeletons. Nine bone fragments and 21 blood samples of claiming relatives were sent to the genetic laboratory for autosomal STR and mitochondrial DNA (mtDNA) analysis, in order to identify the skeletons and return the remains to their relatives.
2. Materials and methods
2.1 Samples processed
Nine 30-year-old bone samples were processed. DNA extraction was performed on 8 tooth pieces and 1 femur fragment.
Twenty-one FTA cards with blood samples from claiming relatives were also processed.
2.2 DNA extraction
2.2.1 Bone samples
All teeth and bone fragments were cleaned with water and neutral detergent. After several washes with distilled water, the samples were decontaminated with 10% commercial bleach and were ground using a cryogenic grinder (Freezer Mill 6750-Spex Centriprep Inc.). The powdered sample (0.4 g) was decalcified 48 h with continuous rotation and then incubated in 1.2 ml of lysis buffer and 60 μl of Proteinase K (20 mg/ml) at 56 °C during 18 h [
[1]
]. Afterwards, DNA extraction was carried out with an equal volume of phenol:chloroform (50:50) twice and then once with chloroform. The aqueous phase was concentrated in 150 μl by using Microcon Centrifugal Filter Unit (Millipore). Finally, the extracted DNA was purified by using QIAamp Blood Mini Kit (Qiagen).2.3 Blood samples
DNA extraction from blood samples on FTA cards was performed by using Proteinase K digestion, phenol:chloroform (50:50) extraction and alcoholic precipitation.
2.4 PCR amplification
With the aim of replicating results and obtaining a consensus profile in the samples analyzed, the following commercial kits were used: AmpFℓSTR® Identifiler® PCR Amplification Kit, AmpFℓSTR® Profiler Plus® PCR Amplification Kit and AmpFℓSTR® COfiler® PCR Amplification Kit (Applied Biosystems), according to the conditions suggested by the manufacturer.
Furthermore, 13 markers were analyzed by using reduced-size STR amplicons (so-called mini-STR) according to: Multiplex 1 (TH01, Amelogenina, FGA, D18S51, D16S539 and D2S1338), Multiplex 2 (D13S317, CSF1PO, D7S818 and D8S1179) and Multiplex 3 (Penta D, Penta E and D21S11) [
2
, 3
, 4
].Fragments were analyzed by capillary electrophoresis by using the ABI PRISM 310 Genetic analyzer. Data were collected by ABI Data Collection software version 3.1.0. Collected data were processed and analyzed by using Genemapper ID v3.2 software.
For the amplification of mtDNA HV1 and HV2 regions, the following primers were used: 15997F, 16068F, 16159F, 16225F, 16096R, 16236R, 16260R, 16401R, 06F, 048F, 130F, 172F, 255F, 205R, 285R, 408R and 430R. By combining different primer sets, fragments of approximately 400 bp (HV1 and HV2), 250 bp (HV1A-HV1B, HV2A-HV2B) and 150 bp (HV1a-HV1b-HV1c; HV2a-HV2b-HV2c) were amplified. For the sequencing reaction, the Big Dye Terminator 3.1 (Applied Biosystems) was used. mtDNA sequence analysis was performed by using Sequencher 4.2 Demo software.
A posterior probability of greater than 99.9% was required to report the identifications [
[5]
].C.H. Brenner, DNA·VIEW™. http://dna-view.com/liography.
3. Results and discussion
A total of 9 bone samples were typed for 15 STR markers and Amelogenin using commercial kits and for13 mini-STRs using 3 multiplexes developed “in-house”. The genetic profiles obtained were classified following these criteria:
- •Complete: Results in 13–15 of the typed markers.
- •Partial: Results in 6–12 of the typed markers.
- •Incomplete: Results in 5 or less of the typed markers.
Complete STR profiles in 7 tooth pieces as well as an incomplete profile in 1 tooth and 1 femur fragment belonging to the same skeleton were obtained (see Table 1). One of the skeletons (sample 58-F-11) had a strong presumptive identification with a patrilineal relative, which is why Y-STRs were typed using AmpFℓSTR® Y-filer® PCR Amplification Kit (Applied Biosystems) in 1 tooth sample and in 1 blood sample. For both samples, complete haplotypes were obtained.
Table 1Samples proceced and results for STR and mtDNA study.
| Skeletons analyzed | Type of bone sample | STR profile | mtDNA amplicon size used |
|---|---|---|---|
| 10-I-23 | Tooth | Complete | 400 bp |
| 57-E-15 | Tooth | Complete | 400 bp |
| 55-J-6 | Tooth | Complete | 400 bp |
| 58-G-28 | Tooth | Complete | 400 bp |
| 56-B-34 | Tooth | Complete | 400 bp |
| 58-F-11 | Tooth | Complete | 400 bp |
| 56-L-12 | Tooth | Complete | 400 bp |
| 52-L-23 | Tooth | Incomplete | 250 bp |
| 52-L-23 | Femur | Incomplete | 250 bp |
For mtDNA sequencing, HV1 and HV2 regions were amplified in 9 bone samples. The sequences obtained were classified by the fragment size that was needed to amplify to get a complete sequence from the sample:
- •400bp: HV1 and HV2.
- •250bp: HV1A, HV1B, HV2A and HV2B.
- •150bp: Not used.
Mitochondrial DNA was sequenced successfully in 7 tooth samples with 400 bp amplicon sized fragments. In 1 tooth sample and 1 femur fragment, it was necessary to use 250 bp amplicon sized fragments to obtain a complete mtDNA sequence.
The use of 150 bp amplification was not necessary for these samples (see Table 1). Fourteen blood samples were amplified and complete sequences were obtained in all of them using 400 bp amplicon sized fragments.
4. Conclusions
The results obtained for the tooth samples showed a high proportion of complete autosomal STR profiles and also of complete mtDNA sequences. These results are consistent with previous experiences in our laboratory (data not shown).
The STR, Y-STR and/or mtDNA data were used depending on the presumptive identification of each skeleton (Table 1, Table 2).
Table 2Relatives and markers involved for the identifications.
| Skeletons identified | Relatives involved for the identification | Useful markers |
|---|---|---|
| 10-I-23 | Mother, brother and sister | STR and mtDNA |
| 55-J-6 | Brother and sister | STR and mtDNA |
| 58-G-28 | Two daughters | STR and mtDNA |
| 58-F-11 | Paternal cousin | Y-STR |
For the particular case of skeleton 58-F-11 there was a strong presumptive identification, since this person was the only male disappeared in the paternal line in that family. The identification of 58-F-11 with a paternal cousin was possible using only the Y-haplotype of both samples.
The skeleton 52-L-23 presented an incomplete STR profile (tooth and femur samples). In this case, the mtDNA sequence could contribute to a future identification.
Conflict of interest
None.
Role of funding
Equipo Argentino de Antropología Forense (EAAF) and LIDMO provided the financial support for this article. The funding source had no involvement in study design.
Acknowledgements
The authors wish to thank Meredith Moon and Marcela Villar for providing language help and writing assistance.
References
- DNAtyping from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples.Croat. Med. J. 2001; 42: 260-266
- Characterization of new MiniSTR loci to aid analysis of degraded DNA.J. Forensic Sci. 2005; 50
- A study on the effects of degradation and template concentration on the amplification efficiency of the STR miniplex primer sets.J. Forensic Sci. 2004; 49
- The development of reduced size STR amplicons as tools for the analysis of degraded DNA.J. Forensic Sci. 2003; 48: 1054-1064
C.H. Brenner, DNA·VIEW™. http://dna-view.com/liography.
Article info
Publication history
Published online: October 05, 2009
Accepted:
August 26,
2009
Received:
August 20,
2009
Identification
Copyright
© 2009 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.
