DNA Mixtures, Forensic Interpretation of Mass Graves

War crimes are most often committed during conflicts between nations. Crimes against humanity, such as summary executions of civilians, are also not uncommon in situations of national armed conflicts, revolutions, or in totalitarian regimes. All of these events result in missing persons and often in undiscovered mass graves. Both are searched for and processed by forensic experts in the aftermath of the conflict. Legal medicine has greatly benefited from the development of molecular biology and its new analytical techniques, in particular DNA analysis, in the identification of highly decomposed human remains.

Terrorist attacks on densely populated areas or against large human gatherings, such as the 2001 attack on the World Trade Center and the 2002 Bali nightclub bombing, or mass disasters, such as the 2004 Indian Ocean earthquake and tsunami, also yield a tragically large amount of bodies to be identified. In some situations, such as after the conflict in Kosovo in the 1990s, the identification of human remains was possible in many cases by comparing medical and dental records of missing persons with findings at autopsy in cadavers rescued from mass graves. Mass graves containing a large amount of highly decomposed bodies and skeletal remains do pose specific challenges for forensic experts, especially when dental and medical records are not available, or when the grave is a secondary one, containing parts of bodies who were purposely removed and mixed by the perpetrators. The recent development of new DNA sequencing and profiling technologies, as well as the understanding of the uniqueness of certain DNA sequences among individuals, has become greatly useful for human identification in situations of mass casualty.

Discrete genetic variations among individuals in the population and among races are known as polymorphisms. The word polymorphism originates from the Greek poly, meaning several and morphos, meaning shape. Single nucleotide polymorphisms (SNP) are mutations of one base pair in the sequence of certain genes. Several polymorphisms are present in regions of DNA known as microsatellites, as well as in some types of repeated DNA sequences. Polymorphisms are of special interest in forensic investigation because they allow the identification of a person or suspect through the DNA extracted from a bloodstain by comparing it with another sample from the suspect, or the identification of a baby's mother or father, or of a murder victim through a bloodstain or saliva left at a crime scene, or the identification of unknown human remains by matching the DNA with that of a living relative. The latter procedure was frequently applied in the aftermath of the Asian tsunami in December 2004.

Most genes are inherited in two copies: one from the father and the other from the mother, which are respectively known as paternal and maternal alleles. Allelic comparison of certain genes is one of the identification techniques used in forensics. Conversely, DNA from cell organelles, the mitochondria, is only inherited from the mother (mtDNA), allowing the comparison of the mtDNA collected from a sibling, or from the mother with the mtDNA of a deceased person, to establish whether those remains belong to that family. The remains of an American soldier who served in Vietnam, for instance, were identified 24 years after his death through the mtDNA extracted from the bone marrow of his skeleton and samples donated from living relatives. Mitochondrial DNA does not identify an individual in particular, however, if more than one sibling is missing. When several members of a family are missing, other genetic and forensic tests may help to determine the identity of the remains found in a disaster area or in a mass grave.

Some specific repeating DNA sequences are also used for human identification. They are classified according to their characteristics, such as LINES (long interspersed sequences), SINES (short interspersed sequences), LTR (long terminal repeats), STR (short tandem repeats), and VNTR (variable number of tandem repeats, or microsatellite DNA). Short tandem repeats are used in tests of paternity and VNTR is used to identify victims and suspects.

Sir Alec Jeffreys, an English professor at Leicester University, was the first scientist to use DNA polymorphism tests in a forensic identity investigation in 1985. He chose a region of DNA known as VNTR because of its great variability (polymorphism) among individuals, which can only yield a perfect sequence match in cases of identical twins. Additionally, if two individuals do present a high degree of similarities (but not a perfect match) in their VNTR sequences, this indicates that they are related.

However, postmortem transformations quickly degrade DNA of soft tissues due to bacterial activity. Hochmeister and colleagues (Journal of Forensic Science, 1991;36:1649–1661) were the first to isolate DNA from the marrow of the human femur from a mummified body of an 11-year-old child and from a corpse that had been submerged in water for 18 months. They utilized two DNA analysis techniques to sequence VNTR from the bones: VNTR amplification by PCR (polymerase chain reaction) and RFLP (restriction fragment length polymorphisms), thus demonstrating that DNA is relatively well preserved in the marrow of long bones. In 1994 another group identified the Romanov family (the last Russian czar, his wife and three children killed in 1918), also using DNA extracted from their bones. Studies conducted by other researchers in 1996 found that even in bones DNA degrades differently in certain conditions, being best preserved in dry or arid environments such as deserts and worst degraded when the bone fragments were immersed in water. Teeth and dried blood stains however, even almost 100 years old, were found to be a good source of well preserved DNA for forensic identification of human remains.

Crime scenes, disaster areas, and mass graves often produce mixed biological samples containing genetic material from two or more individuals, such as mixed body fluids , bloodstains, or blood pools. In the 1994 murder case of Nicole Brown Simpson, for instance, the blood pool around the body of Nicole Simpson contained blood (and DNA) of both Nicole and her former husband, O. J. Simpson, who was charged and acquitted in criminal court and later found liable in civil court for her murder. When male and female cells are present in the same sample, the cells can be sorted by using a laser technique or DNA profiling . In mass graves or bombing scenes however, samples may contain DNA of several victims, rapidly degrading in tropical climates or due to moisture and soil conditions, all of which affect the STR sequences and other DNA loci used in human identification. Until recently, this constituted a serious obstacle to forensic interpretation. With the rapid evolution of genetic screening technologies, however, new test kits and software are being constantly developed. Some of these kits are more sensitive to low, degraded concentrations of DNA found in mixed samples than others.

Additionally, matches from DNA mixtures are also assessed against scientific profiles and probability or frequency estimates in order to establish the statistical significance of the match. These statistics serve to inform courts and jurors of the odds that a match can be common to more than one individual. In degraded DNA mixtures, the margin of error for minor STR components can be 10–33%, and the burial of several relatives with similar alleles in a mass grave can make individual identification of DNA from a mixture even more difficult. Statistical evaluation of DNA mixtures takes into account the match probability.

The International Commission on Missing Persons installed a forensic laboratory in Tuzla, Bosnia, in 2000 for the identification of victims of the war in Bosnia. The forensic team has chosen a more direct approach than DNA mixture analysis. Living relatives of missing persons donate blood samples for comparison with DNA extracted from the long bones and teeth of the exhumed human remains. In Bosnia, more than 30,000 people were reported missing, mostly Muslim men and boys. Mass graves were located and exhumed in nearby Srebrenica and other localities, but many mass graves are yet to be located and processed. To identify one corpse, this method requires samples from 3 relatives. Whenever nuclear DNA is not degraded, it is used in the tests, because the maternal and paternal alleles offer a unique profile for each individual. The other option is mtDNA profiling because mitochondrial DNA is usually better preserved and stays unaltered for longer periods than nuclear material.

ICMP is also profiling DNA from other mass graves in Croatia, Kosovo, and other former Yugoslavia territories. In Rwanda however, where more than 500,000 people were massacred in 3 months in 1994, due to the inexistence of automated DNA technologies and the rapid degradation of DNA because of the tropical heat and humidity, this task would have been impossible. In Brazil, collective summary executions occurred during the totalitarian military regime (1964–1985) and the location of many suspected mass graves remains unknown. Some were discovered in the 1990s. Legal physicians working in the identification of the human remains recovered from mass graves were faced with the same problems of DNA degradation common in other tropical countries. Nevertheless, DNA extracted from teeth and the application of other forensic identification techniques led to the successful identification of many of the victims.

see also Autopsy; Blood; DNA; DNA sequences, unique; DNA typing systems; Exhumation; Gene; Genetic code; Identification of the son of Louis XVI and Marie Antoinette; Laser; Medical examiner; Mitochondrial DNA analysis; Mitochondrial DNA typing; Mummies; Pathology; PCR (polymerase chain reaction); RFLP (restriction fragment length polymorphism); Skeletal analysis; War crimes trials; War forensics; Y chromosome analysis.