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Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains
Forensic Science International: Genetics Supplement Series 2 (2009) 269–270
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains Suni M. Edson a,*, Alexander F. Christensen b, Suzanne M. Barritt a, Audrey Meehan b, Mark D. Leney b,1, Louis N. Finelli a a b
Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Bldg 101, Rockville, MD 20850, United States Joint POW/MIA Accounting Command – Central Identification Laboratory, Hickam Air Force Base, HI 96853, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 September 2009 Accepted 16 September 2009
Sampling of cranial fragments for mitochondrial DNA (mtDNA) analysis is a common practice for identification of skeletonized human remains. The Armed Forces DNA Identification Laboratory (AFDIL) and Joint POW/MIA Accounting Command – Central Identification Laboratory (JPAC-CIL) work in concert to identify the remains of US service members killed in past military conflicts. When dealing with samples taken from remains that are commingled or lack a secure archaeological context, multiple elements must be sampled from the same burial or case. In such cases, the cranium is often fragmentary. Previous work examined the success rate of approximately 4000 skeletal elements for sequencing of mtDNA. The approximately 50% success of the crania seemed anomalous considering the frequency with which they are sampled. Subsequent studies of the 558 cranial fragments tested from 1992 to August 2009 were done to examine the independent rates of success of the portions of the skull. It was found that each yielded reportable mtDNA sequence at rates that exhibited statistically significant differences. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Skeletal sampling Cranial fragments Human identification
1. Introduction In the field of human identification, the effective sampling of osseous remains for DNA analysis will increase the probability of making an identification. When remains are well-preserved, a single sample may suffice; however, multiple samples are generally necessary in cases of mass disasters [4,5], sets of highly commingled remains [6], and groups of remains with a loss of context [7–9]. Since its inception in 1992, the Armed Forces DNA Identification Laboratory (AFDIL) has worked together with the Joint POW/MIA Command – Central Identification Laboratory (JPAC-CIL) to identify the remains of missing United States service members from past military conflicts. Between 2004 and 2005, all elements having been processed up to that date were surveyed for degrees of success at producing a reportable mtDNA sequence [1– 3]. The results were as anecdotally expected: the largest long bones, the femur and tibia, were generally more successful, at 95% and 88%, respectively [1]. Unexpectedly, only 51% of cranial samples were successful. Oftentimes the cranium may be independently identified through analysis of the dentition;
* Corresponding author. Tel.: +1 301 319 0135; fax: +1 301 295 5932. E-mail address: [email protected] (S.M. Edson). 1 Current address: Massachusetts Biologic Laboratories, University of Massachusetts Medical School, Jamaica Plain, MA 02310, United States. 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.09.029
therefore, sampling of the cranium for DNA is key for reassociating post-cranial remains. It became of interest to AFDIL and JPAC-CIL to improve the sampling of this element and to determine the optimal region of the skull to remove for DNA analysis. 2. Methods and materials JPAC-CIL removes a fragment of bone from osseous materials recovered in the field and submits it to AFDIL for mtDNA processing. From 1992 to August 2009, 7712 skeletal elements have been received and processed at AFDIL. From 1992 to 2007, samples were cleaned and extracted using the protocols outlined in Edson et al. [1]. From 2007, a material modification was made to the extraction protocol reducing the volume of bone needed for extraction from 2.0–2.5 g to 0.2 g, and incorporating a demineralization buffer that allows for a complete dissolution of the bone [10]. However, all processing subsequent to extraction follows the amplification and sequencing protocols outlined in Edson et al. [1]. An element was considered to be successful if 100 bp or more of mtDNA sequence was generated and reported. AFDIL reports an mtDNA sequence only if there is confirmation of that sequence from at least two independent amplifications. Success rate data is calculated as a cumulative number across both extraction methods and any other modifications to technique (i.e., implementation of mini-primer sets in 2000 [11]).
270
S.M. Edson et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 269–270
Table 1 Success rates of cranial elements submitted to AFDIL for processing from JPAC-CIL. The p-value is calculated based on the success of the indicated element vs. that of the temporal with an a = 0.05. Portion of skull
Number processed
% Success
p-Value
Temporal Unspecified skull Frontal Occipital Parietal
149 31 40 117 221
90 52 68 65 52
1.9017E 07 0.00039102 7.0668E 07 1.4087E 14
3. Results
Conflict of interest statement
A combined analysis of both extraction methods for the previous 17 years indicates higher success results for the cranium than seen before [1–3], with a 66% success. This can be compared to the 47% success rate for the skull for nuclear DNA sequencing from World Trade Center victims [5]. However, when the different bones of the cranium are separated, there is a statistically significant difference between the rates of success (Table 1). The temporal bone shows a 90% success, which is markedly greater than that of the other cranial bones. The temporal bone is one of the best bones in the body to sample, with a success rate in line with that of the long bones. The present elevated success rate of the combined cranial samples is most likely due to the increase in sampling from the temporal element since 2004.
The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or as views of the US Department of Defense, the US Department of the Army, the US Department of the Navy, the American Registry of Pathology, the Armed Forces Institute of Pathology, or the Armed Forces Medical Examiner System.
4. Discussion Over time, the temporal has not been the most commonly sampled cranial element for the work done at AFDIL and JPAC-CIL (149 temporals vs. 221 parietals). This is largely due to the fact that sampling of the temporal bone from the cranium can be a less than straight-forward process. Rather than removing an external cutting of the temporal region, it is optimal to take the portion of the temporal that is within the skull, more specifically, the petrous portion. This area of the skull is known to be highly resistant to damage and frequently remains intact, even in circumstances with a high degree of fragmentation [12]. It follows that this should be an optimal region from which to recover mitochondrial DNA. Sampling of the temporal region has increased since the initial survey of skeletal element success rates for mtDNA analysis [1–3]. It is now the primary cranial bone sampled (since 2007: 56 temporals vs. the next highest, 23 occipitals). 5. Conclusions When sampling the cranium for mitochondrial DNA analysis, it is optimal to remove the petrous portion of the temporal. Even in circumstances where only a single sample is needed from a set of skeletal remains, the petrous portion should be considered.
Acknowledgements The authors would like to thank all employees, past, present and future, of AFDIL and JPAC-CIL. References [1] S.M. Edson, J.P. Ross, M.D. Coble, T.J. Parsons, S.M. Barritt, Naming the dead— confronting the realities of rapid identification of degraded skeletal remains, Forensic Sci. Rev. 16 (2004) 63–90. [2] S.M. Edson, H.A. Thew, F.E. Damann, C.A. Boyer, S.M. Baritt, B.C. Smith, Success rates for recovering mitochondrial DNA (mtDNA) from 4,000 ‘ancient’ human skeletal remains, IAFS (2005) 58 (Abstract A0606). [3] M.D. Leney, Sampling skeletal remains for ancient DNA (aDNA): a measure of success, Historical Archaeol. 40 (2006) 31–49. [4] A.Z. Mundorff, R.C. Shaler, E. Bieschke, E. Mar-Cash, Marrying anthropology and DNA: Essential for solving complex commingling problems in cases of extreme fragmentation, in: B. Adams, J. Byrd (Eds.), Recovery, Analysis, and Identification of Commingled Human Remains, Humana Press, Totowa, 2008, pp. 285–300. [5] A.Z. Mundorff, E.J. Bartelink, E. Mar-Cash, DNA preservation in skeletal elements from the World Trade Center disaster: recommendations for mass fatality management, J. Forensic Sci. 54 (2009) 739–745. [6] Sˇ. Anpelinovic´, D. Sutlovic´, I.E. Ivkosˇic´, V. Sˇkaro, A. Ivkosˇic´, F. Paic´, B. Rezˇic´, M. Definis-Gojanovic´, D. Primorac, Twelve-year experience in identification of skeletal remains from mass graves, Croat. Med. J. 46 (2005) 530–539. [7] S. Edson, Identifying missing US servicemembers from the Korean War—do storage conditions affect the success rate of mtDNA testing? Profiles DNA 10 (2007) 14–15. [8] K. Nelson, T. Melton, Forensic mitochondrial DNA analysis of 116 casework skeletal samples, J. Forensic Sci. 52 (2007) 557–561. [9] F.D. Damann, S.M. Edson, Blending anthropology and molecular biology: case studies in sorting and identifying remains of US war dead, in: B. Adams, J. Byrd (Eds.), Recovery, Analysis, and Identification of Commingled Human Remains, Humana Press, Totowa, 2008, pp. 301–315. [10] O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Gen. 1 (2007) 191–195. [11] M.N. Gabriel, E.F. Huffine, J.H. Ryan, M.M. Holland, T.J. Parsons, Improved mtDNA sequence analysis of forensic remains using a ‘‘mini-primer’’ set amplification strategy, J. Forensic Sci. 46 (2001) 247–253. [12] J.K. Kalmey, T.A. Rathbun, Sex determination by discriminant function analysis of the petrous portion of the temporal bone, J. Forensic Sci. 41 (1996) 865–867.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains Suni M. Edson a,*, Alexander F. Christensen b, Suzanne M. Barritt a, Audrey Meehan b, Mark D. Leney b,1, Louis N. Finelli a a b
Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Bldg 101, Rockville, MD 20850, United States Joint POW/MIA Accounting Command – Central Identification Laboratory, Hickam Air Force Base, HI 96853, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 September 2009 Accepted 16 September 2009
Sampling of cranial fragments for mitochondrial DNA (mtDNA) analysis is a common practice for identification of skeletonized human remains. The Armed Forces DNA Identification Laboratory (AFDIL) and Joint POW/MIA Accounting Command – Central Identification Laboratory (JPAC-CIL) work in concert to identify the remains of US service members killed in past military conflicts. When dealing with samples taken from remains that are commingled or lack a secure archaeological context, multiple elements must be sampled from the same burial or case. In such cases, the cranium is often fragmentary. Previous work examined the success rate of approximately 4000 skeletal elements for sequencing of mtDNA. The approximately 50% success of the crania seemed anomalous considering the frequency with which they are sampled. Subsequent studies of the 558 cranial fragments tested from 1992 to August 2009 were done to examine the independent rates of success of the portions of the skull. It was found that each yielded reportable mtDNA sequence at rates that exhibited statistically significant differences. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Skeletal sampling Cranial fragments Human identification
1. Introduction In the field of human identification, the effective sampling of osseous remains for DNA analysis will increase the probability of making an identification. When remains are well-preserved, a single sample may suffice; however, multiple samples are generally necessary in cases of mass disasters [4,5], sets of highly commingled remains [6], and groups of remains with a loss of context [7–9]. Since its inception in 1992, the Armed Forces DNA Identification Laboratory (AFDIL) has worked together with the Joint POW/MIA Command – Central Identification Laboratory (JPAC-CIL) to identify the remains of missing United States service members from past military conflicts. Between 2004 and 2005, all elements having been processed up to that date were surveyed for degrees of success at producing a reportable mtDNA sequence [1– 3]. The results were as anecdotally expected: the largest long bones, the femur and tibia, were generally more successful, at 95% and 88%, respectively [1]. Unexpectedly, only 51% of cranial samples were successful. Oftentimes the cranium may be independently identified through analysis of the dentition;
* Corresponding author. Tel.: +1 301 319 0135; fax: +1 301 295 5932. E-mail address: [email protected] (S.M. Edson). 1 Current address: Massachusetts Biologic Laboratories, University of Massachusetts Medical School, Jamaica Plain, MA 02310, United States. 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.09.029
therefore, sampling of the cranium for DNA is key for reassociating post-cranial remains. It became of interest to AFDIL and JPAC-CIL to improve the sampling of this element and to determine the optimal region of the skull to remove for DNA analysis. 2. Methods and materials JPAC-CIL removes a fragment of bone from osseous materials recovered in the field and submits it to AFDIL for mtDNA processing. From 1992 to August 2009, 7712 skeletal elements have been received and processed at AFDIL. From 1992 to 2007, samples were cleaned and extracted using the protocols outlined in Edson et al. [1]. From 2007, a material modification was made to the extraction protocol reducing the volume of bone needed for extraction from 2.0–2.5 g to 0.2 g, and incorporating a demineralization buffer that allows for a complete dissolution of the bone [10]. However, all processing subsequent to extraction follows the amplification and sequencing protocols outlined in Edson et al. [1]. An element was considered to be successful if 100 bp or more of mtDNA sequence was generated and reported. AFDIL reports an mtDNA sequence only if there is confirmation of that sequence from at least two independent amplifications. Success rate data is calculated as a cumulative number across both extraction methods and any other modifications to technique (i.e., implementation of mini-primer sets in 2000 [11]).
270
S.M. Edson et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 269–270
Table 1 Success rates of cranial elements submitted to AFDIL for processing from JPAC-CIL. The p-value is calculated based on the success of the indicated element vs. that of the temporal with an a = 0.05. Portion of skull
Number processed
% Success
p-Value
Temporal Unspecified skull Frontal Occipital Parietal
149 31 40 117 221
90 52 68 65 52
1.9017E 07 0.00039102 7.0668E 07 1.4087E 14
3. Results
Conflict of interest statement
A combined analysis of both extraction methods for the previous 17 years indicates higher success results for the cranium than seen before [1–3], with a 66% success. This can be compared to the 47% success rate for the skull for nuclear DNA sequencing from World Trade Center victims [5]. However, when the different bones of the cranium are separated, there is a statistically significant difference between the rates of success (Table 1). The temporal bone shows a 90% success, which is markedly greater than that of the other cranial bones. The temporal bone is one of the best bones in the body to sample, with a success rate in line with that of the long bones. The present elevated success rate of the combined cranial samples is most likely due to the increase in sampling from the temporal element since 2004.
The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or as views of the US Department of Defense, the US Department of the Army, the US Department of the Navy, the American Registry of Pathology, the Armed Forces Institute of Pathology, or the Armed Forces Medical Examiner System.
4. Discussion Over time, the temporal has not been the most commonly sampled cranial element for the work done at AFDIL and JPAC-CIL (149 temporals vs. 221 parietals). This is largely due to the fact that sampling of the temporal bone from the cranium can be a less than straight-forward process. Rather than removing an external cutting of the temporal region, it is optimal to take the portion of the temporal that is within the skull, more specifically, the petrous portion. This area of the skull is known to be highly resistant to damage and frequently remains intact, even in circumstances with a high degree of fragmentation [12]. It follows that this should be an optimal region from which to recover mitochondrial DNA. Sampling of the temporal region has increased since the initial survey of skeletal element success rates for mtDNA analysis [1–3]. It is now the primary cranial bone sampled (since 2007: 56 temporals vs. the next highest, 23 occipitals). 5. Conclusions When sampling the cranium for mitochondrial DNA analysis, it is optimal to remove the petrous portion of the temporal. Even in circumstances where only a single sample is needed from a set of skeletal remains, the petrous portion should be considered.
Acknowledgements The authors would like to thank all employees, past, present and future, of AFDIL and JPAC-CIL. References [1] S.M. Edson, J.P. Ross, M.D. Coble, T.J. Parsons, S.M. Barritt, Naming the dead— confronting the realities of rapid identification of degraded skeletal remains, Forensic Sci. Rev. 16 (2004) 63–90. [2] S.M. Edson, H.A. Thew, F.E. Damann, C.A. Boyer, S.M. Baritt, B.C. Smith, Success rates for recovering mitochondrial DNA (mtDNA) from 4,000 ‘ancient’ human skeletal remains, IAFS (2005) 58 (Abstract A0606). [3] M.D. Leney, Sampling skeletal remains for ancient DNA (aDNA): a measure of success, Historical Archaeol. 40 (2006) 31–49. [4] A.Z. Mundorff, R.C. Shaler, E. Bieschke, E. Mar-Cash, Marrying anthropology and DNA: Essential for solving complex commingling problems in cases of extreme fragmentation, in: B. Adams, J. Byrd (Eds.), Recovery, Analysis, and Identification of Commingled Human Remains, Humana Press, Totowa, 2008, pp. 285–300. [5] A.Z. Mundorff, E.J. Bartelink, E. Mar-Cash, DNA preservation in skeletal elements from the World Trade Center disaster: recommendations for mass fatality management, J. Forensic Sci. 54 (2009) 739–745. [6] Sˇ. Anpelinovic´, D. Sutlovic´, I.E. Ivkosˇic´, V. Sˇkaro, A. Ivkosˇic´, F. Paic´, B. Rezˇic´, M. Definis-Gojanovic´, D. Primorac, Twelve-year experience in identification of skeletal remains from mass graves, Croat. Med. J. 46 (2005) 530–539. [7] S. Edson, Identifying missing US servicemembers from the Korean War—do storage conditions affect the success rate of mtDNA testing? Profiles DNA 10 (2007) 14–15. [8] K. Nelson, T. Melton, Forensic mitochondrial DNA analysis of 116 casework skeletal samples, J. Forensic Sci. 52 (2007) 557–561. [9] F.D. Damann, S.M. Edson, Blending anthropology and molecular biology: case studies in sorting and identifying remains of US war dead, in: B. Adams, J. Byrd (Eds.), Recovery, Analysis, and Identification of Commingled Human Remains, Humana Press, Totowa, 2008, pp. 301–315. [10] O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency DNA extraction from bone by total demineralization, Forensic Sci. Int. Gen. 1 (2007) 191–195. [11] M.N. Gabriel, E.F. Huffine, J.H. Ryan, M.M. Holland, T.J. Parsons, Improved mtDNA sequence analysis of forensic remains using a ‘‘mini-primer’’ set amplification strategy, J. Forensic Sci. 46 (2001) 247–253. [12] J.K. Kalmey, T.A. Rathbun, Sex determination by discriminant function analysis of the petrous portion of the temporal bone, J. Forensic Sci. 41 (1996) 865–867.