Throughout United States history, the military services have to the best of their ability attempted to recover and identify it's deceased military personnel. In 1981, President Reagan placed the issue of accounting for American servicemembers from Southeast Asia as a matter of highest national priority. This position has been reaffirmed by all Presidents since. The Department of Defense has been tasked to investigate and account, to the greatest extent possible, for the "unaccounted for" Americans and repatriate, identify, and return the remains to their families. Today, there are over 2,200 servicemembers from Southeast Asia, 132 servicemembers from the Cold War era, and over 8,100 servicemembers from Korea, whose remains have not been recovered and/or identified (POW/MIA Fact Book, Department of Defense, October 1992).

The United States Army Central Identification Laboratory, Hawaii (CILHI) of the U.S. Army Casualty and Memorial Affairs Operation Center (CMAOC) is responsible for the recovery, identification, and processing of human remains from previous conflicts. CILHI uses traditional forensic odontological and anthropological methods, as well as other state of the art methods to identify human remains, including the Computer Assisted Post Mortem Identification (CAPMI) dental system and computerized craniofacial superimposition. CILHI uses state of the art photographic, microscopic, and radiographic equipment to accomplish their mission. The availability of records, the passage of time, and the environment to which remains have been exposed are obstacles to traditional identification efforts. Emerging technologies offer new opportunities.

In 1991, the Army contacted the Armed Forces DNA Identification Laboratory (AFDIL), a division of the office of the Armed Forces Medical Examiner (OAFME) at the Armed Forces Institute of Pathology (AFIP) to apply mitochondrial DNA (mtDNA) technology to

the identification of human remains. The mtDNA analysis and other corroborating evidence has since successfully identified the remains of Americans recovered in Southeast Asia. This technology now offers the prospect of identifying remains in the absence of name association, dental, medical, fingerprint, or circumstantial evidence.

The AFDIL has been in the world-wide vanguard of activities to identify remains through the use of DNA technology, and thus carries on the fine tradition of military biomedical research. From the first command-directed immunization program, inoculation for smallpox in President Washington's Army, up to and including the present time, many military and civilian medical scientists continue to make seminal contributions to military and general medicine. Among the many contributions, are Beaumont's studies of digestion in 1824; the founding of the first American School of Preventive Medicine and Public Health in 1893; and Walter Reed's proof that mosquitoes transmit yellow fever in 1900; antimalarial drugs such as mefloquine halotantrine; vaccines such as VEE, typhoid, hemorrhagic fever, adenovirus and meningococcus; plasma and albumin blood products, and CPDA-1, AS-1 and AS-3 blood preservatives. The fields of burn therapy and emergency medicine have their roots in the military and are largely patterned after developments in military medicine. Through the Advanced Research Projects Agency, the military funded efforts that led to the development of the CAT scan and MRI. (Dora Strother, Army Science Board, 50 Years of Accomplishments in Army Research and Development, Social & Scientific Systems, Inc. Bethesda, MD)

The benefit of this military initiative and leadership can now be seen in the specialized area of DNA analysis of human remains. The use of mtDNA identification on remains by AFDIL continues that legacy of pioneering advances in medicine that contribute to society at large. This national resource can be used to assist in the identification of remains not only of servicemen and women who died in battle, but also to other disaster related deaths such as aircraft mishaps, earthquakes, explosions, and fires.

Despite difficulties in extracting mtDNA from ancient remains, the AFDIL has been successful in positively correlating mtDNA extracted from skeletal remains to their maternal relatives in

many CILHI cases. In addition, the AFDIL has performed the mtDNA sequence analysis on the skeletal remains and awaits family blood reference specimens for comparison in numerous other CILHI cases.

In one case, "X-611, conflicting results were received from the mtDNA analysis of remains analyzed by the AFDIL and a laboratory of the University of California-Berkeley. The source of the discrepancy has not been determined. This discrepancy case caused the Department of Defense to re-examine the use of mtDNA and to take several actions.

First, uncertainty concerning the efficacy of mtDNA technology created by the discrepancy in case "X-611 caused the U.S. Army on February 3, 1994, to suspend using mtDNA to determine the identity of war remains without corroborating evidence. The Department of Defense supported that position.

Second, the Army recommended that the Department of Defense Science Board (DSB) establish a task force to examine the issues associated with using mtDNA to identify remains. In May 1994, the Defense POW/MIA Office (DPMO) accepted responsibility to be the Department of Defense sponsor for this Task Force. On June 20, 1994, the Under Secretary of Defense requested the Chairman of the Defense Science Board establish a Task Force on the use of DNA technology for identification of ancient remains. The issues (Annex A) for the members of the DSB Task Force (Annex B) were incorporated into a set of Terms of Reference (TOR).

Third, the military reviewed the standards used to perform mtDNA testing and noted that a set of formally recognized and widely accepted technical and quality assurance standards did not exist specifically for mtDNA testing of ancient skeletal remains. It has become well-understood that minuscule levels of contaminants can lead to erroneous results and that extraordinary measures of quality control are needed. The ASD (HA), with input from the forensic and genetics communities, developed a quality assurance program for the Department of Defense that would have credibility and acceptability within the scientific and legal communities, to the families, and to the general public (Annex C).

Fourth, portions of the contested remains were sent to the British Forensic Science Service (FSS, also known as the British Home Office) for analysis. Results confirmed AFDIL's findings

(Annex D). Furthermore, when subsequently and unknowingly challenged by CILHI with skeletal remains of the same case, the AFDIL twice more obtained the same mtDNA sequence result.

On January 6, 1995, the Army concluded that they had confidence in the AFDIL testing results. The Department of Defense then gave approval to respond to all Army requests for analysis and to consider that Army requests have had a waiver, at Army level, to the Army imposed moratorium (Annex E).

Meanwhile, the issue of repatriation and identification of remains from the Korean conflict has come to the forefront. on August 24, 1993, the Korean People's Army (KPA) signed an agreement with the United Nations Command (UNC) for cooperation in the recovery, repatriation, and identification of UNC remains located north of the Demilitarized Zone (DMZ). Since 1990, the North Koreans have repatriated 208 coffins containing purported American remains. Because of the condition of these remains and the paucity of relevant personal, medical, and dental records for servicemembers serving in the Korean action, mtDNA analysis offers the best prospect to identify these remains.

The TOR represent the issues that needed to be addressed before the military proceeded with mtDNA testing of skeletal remains, particularly "unassociated" Korean remains. Paramount is assurance that the technology is cost effective and that families can depend on the methods of identification used.

I. FEASIBILITY

DSB TOR: To determine the feasibility of using DNA techniques for identification of ancient remains as evidenced, in part, by success in identification efforts thus far. [Is the conceptual basis for mtDNA identification of ancient skeletal remains workable? is the discriminatory potential of mtDNA as currently obtained, and with or without other identification data, sufficient for individuation of skeletal remains from Southeast Asia and Korea?]

Nuclear DNA typing has the capacity to be used for identification because DNA is different among all individuals, except identical twins. The potential exists for DNA tests to provide identifications which cannot be made in any other way. Any portion of skeletal remains could potentially be useful for DNA

identification. Since reference specimens for DNA comparison can be obtained from family members, it can be useful in situations even though premortem specimens are not available. In contrast, premortem records must be available for conventional identification methods using medical, dental or anthropological comparisons.

A. Forensic DNA Identification

Molecular techniques have revolutionized the biological sciences. Procedures for rapid DNA sequencing were developed in the 1970s, the Southern blot technique for DNA fragment sizing was developed a few years later, and the polymerase chain reaction (PCR) for DNA fragment amplification in 1985. These techniques have become well established and now are at the heart of innumerable research efforts in the biologic sciences. Molecular biologic techniques have long since moved from the research laboratory to the clinical service laboratory. The revolution created by this new technology has spread to the forensic sciences where DNA typing is taking its place alongside fingerprinting in terms of its impact on the criminal justice system. The basic molecular biologic principles are at this point well established and documented.

The Office of Technology Assessment released its report on the forensic uses of DNA typing in 1990. They concluded that "no scientific doubt remains that technologies already available can accurately detect genetic differences between humans." Similarly, the National Research Council (NRC) of the National Academy of Sciences issued a report in 1992, confirming the capability of DNA testing as a new and important technology to identify the origin of biologic trace evidence. Traditional serologic testing is based on genetic differences that are best characterized at the DNA level.

Courts of law have generally embraced the new DNA technology. The passage of the DNA Identification Act as a part of the 1994 Crime Bill by Congress, to spur creation of a national network of state DNA databases of convicted sex offenders and other felons, is a recognition of the value and validity of this DNA identification technology.

Application of DNA typing to the identification of human remains is obvious. Identification of tissue origin is being performed by many crime labs around the world. The AFDIL has been a leading laboratory devoted to the identification of human remains, and assisting other Federal Government agencies.

However, most DNA identification efforts have thus far focused on the typing of nuclear DNA of relatively recent vintage. Identification of ancient skeletal remains through mtDNA sequencing presents new issues which have not been a significant part of the larger discussions of the application of molecular biology to forensic identification.

B. MTDNA Sequence Identifications

Dr. Mary-Claire King first employed mtDNA to identify the Argentina "disappeared". In these cases, Dr. King would match the mtDNA sequence of children, whose parents were killed for political reasons, to that of purported maternal grandmothers. In 1991, Dr. Mark Stoneking described the use of mtDNA sequencing for the identification of a skull found in the Mojave desert, approximately four years after a 3 year-old girl was reported missing. In 1994, Dr. Peter Gill described the use of mtDNA to identify the Russian Romanov Tsar Nicholas II and his family.

In 1991, the AFDIL first successfully employed mtDNA sequencing to identify the skeletal remains of a servicemember killed in the Southeast Asian conflict; a report of this case was published in the Journal of Forensic Sciences in 1993. Subsequently, AFDIL has performed testing which has led to other identifications.

C. Mitochondria

Human mitochondria are thought to have evolved through incorporation of an intracellular symbiont (parasite) into early life forms (Lynn Margulis, Symbiosis and Cell Evolution, 1981, Freeman Publishing). This intracellular symbiont, similar to a primitive bacterium, had its own DNA. The symbiont flourished within the cytoplasm in harmony with the host cell. Mitochondrial symbionts would pass into the daughter cells of every dividing cell. In time, cells came to depend on the efficient energy utilization mechanisms of this symbiont. This theory of endosymbiotic origin explains many of the peculiar features of this intracytoplasmic organelle.

Mitochondria are the primary means of oxidative respiration of the cell. They are critical to the utilization of oxygen from the air to generate usable energy in the form of phosphorylated compounds for the cell. Hence, mitochondria are considered to be the "powerhouses of the cell".

D. Mitochondrial DNA

Human mtDNA is a circular DNA "particle", 16,569 base pairs in length. The complete sequence from a composite of individuals was published in the journal Nature in 1981 by Anderson, et. al. An MboI restriction site within the major noncoding region was arbitrarily designated as the origin, and the base pairs are numbered sequentially proceeding clockwise (Figure 1). This published sequence is by convention used as a reference sequence in studies of human mtDNA variation, with polymorphisms usually indicated as differences from this reference sequence.

The mtDNA genome contains 37 genes, including 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes (Figure 2). The protein-coding genes include two ATP synthetase subunits, seven NADH dehydrogenase subunits, three cytochrome oxidase subunits, and cytochrome b. These proteins are all involved in electron transport and cellular respiration, the primary function of the mitochondria. All of the remaining several hundred proteins necessary for mitochondrial function (including those required for replication, transcription, and translation of mtDNA) are encoded in the nucleus, and hence must be imported from the cytoplasm.

The two complementary single DNA strands that comprise the double-stranded human mtDNA genome have an asymmetric distribution of guanine and thymine residues, and can be separated as heavy (H) and light (L) strands via ultracentrifugation. Most of the mtDNA genes are transcribed from the H strand, with only one protein-coding gene and eight tRNA genes transcribed from the L strand.

One of the most striking features of the human mtDNA genome is the extreme paucity of noncoding sequence. only 7% does not encode proteins, rRNA, or tRNA, and the coding regions do not contain intervening sequences. By contrast, it is estimated that at least 90% of the nuclear DNA genome is noncoding, and large

and frequent intervening sequences are the rule for nuclear genes. Intergenic regions in mtDNA are usually less than 10bp in length, and for some of the genes polyadenylation of the mRNA transcript is required to form the termination codon. Approximately 90% of the noncoding mtDNA consists of the control region (displacement loop or D-loop), an 1,100 base pairs region that includes the H-strand origin of replication and origins of transcription for both strands. The L-strand origin of replication is located in a noncoding segment of 31 base pairs, located about 5,700 base pairs from the control region.

Cells typically have several hundred to several thousand copies of DNA, each with one to ten mtDNA molecules, whereas most nuclear genes exist in just a single paired complement per cell. Thus, for ancient remains where there may be very small amounts of surviving DNA that is highly degraded, the probability of obtaining a DNA type is greater for mtDNA. For some types of remains, such as telogen (shed) hairs or keratinized skin, nuclear DNA appears to be absent while mtDNA is still present.

Human mtDNA is strictly maternally inherited. Accordingly, the DNA in mitochondria is not present as pairs of genes, one maternal and one paternal, as is the nuclear DNA of chromosomes. Thus, despite the high number of copies, only a single sequence is found and recombinational events do not occur.

Spermatozoa have about 50 to 100 mitochondria in the midpiece, which provides the energy for the spermatozoa to swim. While the midpiece does penetrate the egg upon fertilization, it is not clear what subsequently happens to the paternal mitochondria. It may be that the paternal mitochondria are preferentially sequestered and destroyed. Prior to fertilization the maternal mitochondria increase to about 100,000 to 200,000 in the oocyte (egg). Maternal inheritance may simply reflect this greatly enhanced abundance of maternal mtDNA relative to paternal mtDNA in the egg. In addition, a bottleneck theory has been proposed in which only a few copies of the oocyte mtDNA are actually replicated, excluding the paternal copies from proliferating. Some studies have reported a low level of paternal mtDNA inheritance in Drosophila flies and in mice on the order of 0.01 to 0.001% per generation. However, these studies utilized interspecies crosses and could therefore reflect the peculiar nature of the hybrids. Regardless of the mechanism involved, no exception to maternal inheritance has ever been reported in

humans.

In the absence of new mutations, maternally-related individuals should have identical mtDNA types. For the purposes of individual identification, any maternal relative can therefore serve as a reference. The maternal line includes biological mothers, siblings, maternal aunts and uncles, children of sisters, and children in the case of an unidentified deceased female servicemember. The family relation can be distant. For example, fifth generation maternal relatives were used successfully to identify the remains of the last Tsar of Russia and the Royal family.

Critical for identification purposes, mtDNA is highly polymorphic, differing between most individuals.

E. Polymorphisms

The highly streamlined nature of the vertebrate mtDNA genome initially led to the expectation that it would be highly conserved evolutionarily. In the late 1970's it was postulated that mtDNA evolves, on average, 5-10 times more rapidly than nuclear DNA. Further studies in the laboratories of Dr. Douglas Wallace and Dr. Allan Wilson showed that there were high levels of mtDNA polymorphism within humans. These studies were based on analyses of restriction fragment length polymorphisms (RFLPS) across the entire human mtDNA genome. RFLP analyses have continued to be a valuable source of information concerning human mtDNA variation, evolution, and disease. However, for individual identification purposes, attention has focused instead on DNA sequence analysis of the control region which is known as the displacement loop or D-loop (Figures 3 and 4).

The control region is the major noncoding segment of human mtDNA, and is the most polymorphic segment as well. It is a region of greater than one thousand base pairs (16,024-576), which is instrumental in the regulation and initiation of synthesis of the gene products and replication of the mtDNA. Elsewhere, the mtDNA sequence is highly conserved. Consequently, the level of polymorphism in the coding region is about one third that of the

control region.

Sequence analysis of the entire control region demonstrates that variability is not distributed at random, but rather is concentrated in two hypervariable (HV) segments of about 400 base pairs each, with the first segment (HV1) having about twice as much variability as the second segment (HV2) (Figures 5 and 6). Virtually all subsequent studies of control region sequence variation have therefore analyzed either HV1 alone or both HV1 and HV2.

These studies have revealed much higher levels of mtDNA sequence variation than nuclear DNA sequence variation. Dr. Peter Gill of the Forensic Science Service, sequenced HV1 and HV2 from 100 British Caucasians and found the level of mtDNA nucleotide diversity to be 1.1%, while the level of nuclear DNA nucleotide diversity has been estimated to be at most about 0.1l%; thus, the amount of mtDNA nucleotide diversity in the control region is about ten times the amount of nuclear DNA nucleotide diversity.

It is not clear why mtDNA evolves so rapidly. The rate of evolution is a function of the rate at which new mutations arise, and the rate at which mutations become fixed; there is reason to suspect that both of these factors are elevated for mtDNA. Highly mutagenic by-products of respiration, such as free radicals, are known to be present in mitochondria, and while some repair of mtDNA damage apparently does occur, it does not seem to be as efficient as DNA repair in the nucleus. The rate at which mutations become fixed in a population is inversely related to the effective size of the population, and because of the maternal inheritance of mtDNA, it has a smaller effective size than nuclear DNA. Still, further work is required to understand how mtDNA mutations arise and spread before a general theory relating mtDNA polymorphism, mutation rates, and evolutionary rates can be developed.

F. Ancient DNA

In order for DNA to be useful to the identification of individuals, the DNA must remain sufficiently intact and capable of extraction.

A new field of scientific endeavor has emerged which seeks to extract and amplify DNA from very old biological materials, such