Departments of Genome Sciences and Medicine, School of Medicine, University of Washington, Seattle, WA 981957720, USA.
Keywords Human rights, forensics, DNA, genomics, sequencing, mitochondria, Argentina, Vukovar, Ovcara, Balkans
Accepted 11 October 2001
Dedication
Like everyone else who is a part of the celebration of the 80th year of Mervyn Susser and Zena Stein, the invitation to include my work in this collection has delighted, honoured and overwhelmed me. I have known Zena and Mervyn since I was 30, both my entire life as an independent scientist and my entire life as a mother. That I have been able to be both has been due to the example of Zena, more than anyone in the world. For 25 years, she has supported me intellectually and emotionally, with intercontinental collaborative projects or a cup of tea, whichever was more critical at the moment.
As my birthday present for Mervyn and Zena, I offer the story of genomic sequencing in the service of human rights, because it grows from the way they do science. Not that this is literally a Susser-Stein project. I have been swept up in many of those, but this is not one of them. Rather, I was able to conceive of this project 17 years ago and carry it out since because of the principles I have learned from these two. In thinking about this celebration, I have tried to formulate those principles explicitly, which is a little difficult, because for me, learning from Zena and Mervyn is entirely deductive. But here are a few things I have learned from them that have been important to me:
The most righteous projects demand the most rigorous science.
No question is too big to ask.
The most important questions come from people on the frontlines.
Good and evil are both real, and the distinction is obvious.
Speaking a language poorly is better than not speaking it at all.
Progressive politics are perfectly compatible with good wine.
So many thanks, Mervyn and Zena, and happy birthday. I hope you will like this story.
Mary-Claire King
Tools of genomic analysis have been used to assist the identification of victims of human rights violations.14 Here we describe two recent applications, the identification of a young adult Argentinian born in captivity 22 years ago when his mother was abducted and disappeared in Buenos Aires in 1978, and the identification of remains found in mass graves in the Balkans in the 1990s. In both these situations we used DNA sequences to identify one individual among many (one child among many children; one body among many bodies) by genetically matching the individual to family members whose relative had disappeared.
Identical genomic information is carried by all tissues of a person, and thus is an inseparable source of personal identity. There are two genomes: nuclear and mitochondrial. Nuclear genes are inherited from both parents (Figure 1A); mitochondrial genes are inherited only maternally (Figure 1B
). For resolving questions of identity in human rights contexts, mitochondrial DNA (mtDNA) is especially powerful, for several reasons. First, one purely maternal relative can provide information about an entire lineage. This is possible because a mother transmits her mtDNA sequence to all her children, male and female. Therefore, each person shares their mtDNA sequence with their mother, sisters, brothers, maternal aunts and uncles, maternal grandmother and her brothers and sisters, and so on. In many human rights investigations, only a few relatives survive to provide a record of genetic information. If the missing person (male or female) has one surviving relative to whom he/she is purely maternally related (i.e. related entirely through mothers), then mtDNA can be useful.3 Second, mtDNA is powerful for identification because mtDNA is extremely variable from family to family. Throughout human evolution, and indeed earlier, mutations have accumulated in mtDNA.5,6 Because one portion of the mtDNA genome does not encode any genes, a very large number of mutations could accumulate in this region without any deleterious effect.7 In consequence, one portion of the mtDNA genome is the most variable region of the entire human genome.8 Thus, mtDNA sequences are shared by purely maternal relatives, and each maternal lineage has an mtDNA sequence that is nearly, or even completely, unique. Third, there are many more copies of the mtDNA genome than of the nuclear genome in a cell, so mtDNA is easier to obtain from remains of victims.
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Methods
For all living individuals, small blood samples are obtained by venepuncture or finger prick and blood absorbed on sterile filter paper and sealed in a sterile plastic envelope. Samples are obtained from all participating family members of a missing person. For cases involving living, putatively kidnapped children, blood samples from these children are collected in the same manner at the request of the overseeing courts. For remains of deceased people, any tissue could in principle provide DNA for identification. For remains that are largely skeletonized, as are usually encountered in human rights investigations, teeth or bone are generally the only remaining sources of DNA. In our experience, teeth are the best source of DNA from skeletonized remains, because enamel protects the pulp of the tooth, where cells containing DNA are located, from both degradation and contamination.3 For these projects, DNA was extracted and mtDNA sequenced as previously described.2,3
Although most mtDNA sequences are unique to one lineage, some are public sequences, in that they appear in multiple families who are not closely related to one another. Frequencies of these public mtDNA sequences vary between populations.9,10 For this reason, for each of our projects, it was essential to establish a database of mtDNA sequences obtained from the local population. From the mtDNA sequences of these population controls, we estimate the frequency of each sequence we encounter in an identification case and thereby evaluate the likelihood of observing a match between a missing person and survivors by chance rather than due to biological relationship.
Results
Argentina
In March 1976, a military dictatorship overthrew the elected government of Argentina. Between 1976 and 1983, thousands of Argentinian citizens (and other nationals) disappeared. Among the disappeared were infants and young children, who were kidnapped at the same time as their parents. Also among the disappeared were children born in captivity to young women who were pregnant at the time of their abduction. In 1977, the grandmothers of these children formed the Asociación de Abuelas de Plaza de Mayo to locate the missing grandchildren. The grandmothers collected reports of clandestinely adopted children and undertook a variety of strategies to locate missing children.1,4,11 After the military withdrew from power following defeat in the Falklands War, elections were held and a National Commission on the Disappearance of Persons (CONADEP) was established to determine the identity and circumstances of the disappearances. By September 1984 CONADEP had documented evidence of the kidnapping or killing of over 8800 people.12 By 1986, Nunca Mas, the official report of CONADEP, indicated the actual number of disappeared might be as many as 30 000.13 Included among these were 220 documented cases of disappeared infants and children.
Throughout this period, the grandmothers learned of children who might have been among the kidnap victims. But which children belonged to which biological family? Beginning in 1984, we used genetic testing to identify these children, establishing at the same time a databank of DNA and a database of mtDNA sequences of family members seeking a disappeared child.1,2,4 The Argentinian National Genetic Databank includes one mtDNA sequence representing each participating maternal lineage. We found that mtDNA sequences are quite diverse in the Argentine population. In our database, the average number of nucleotide differences between mtDNA sequences of two randomly selected unrelated individuals is 11.2. This diversity has proven useful in identifying children located by the grandmothers or who have came forward themselves.
The case of Guillermo is a recent example. In 1999, the Abuelas de Plaza de Mayo were informed anonymously of a young man (Guillermo) who might have been an abducted child. Guillermo was interested in participating in the investigation and provided a blood sample to the laboratory. We sequenced mtDNA from Guillermo's sample and compared it to all sequences in our database of Argentinian families. Guillermo's mtDNA sequence was identical to that of one person in our database, namely Rosa, the mother of Patricia (Figure 2).
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As an additional test of the sequence match of Guillermo and Rosa, we obtained a blood sample from an undisputed daughter of Patricia and Jose. This daughter, Mariana, was visiting a friend at the time of her parents' disappearance and hence survived. As anticipated, the mtDNA sequences of Guillermo, Rosa, and Mariana were identical and differed from all other mtDNA sequences in our database. Because our mtDNA database includes >2000 entries, an estimate of the frequency in the Argentinian population of Rosa's sequence is 1/2000. Therefore the likelihood that Guillermo shares this sequence with Rosa (and Mariana) by chance, rather than because they are maternally related) is 1/2000 or 0.0005.
As Guillermo's case indicates, mtDNA sequencing allows identification of a maternal lineage even when information from the family is incomplete. This is the case for the disappeared grandchildren, who are now young adults and whose parents remain missing. More than 60 have been identified and been reunited with their biological families. Guillermo, the most recent, now grapples with the news of his origins.
Croatia
In mass graves remains from many people are co-mingled. Identifying remains present in a mass grave is important both for families and for prosecution of war crimes. Positive identification of individuals can be difficult using only classical forensic analysis. Individuals may swap or hide identifying documents in attempts to flee persecution. Genetic evidence can aid such identifications, adding a level of resolution not previously available.
On 19 November 1991 patients, staff and local refugees were awaiting evacuation from Vukovar hospital in Croatia when Serbian troops overran the hospital. Reports indicate that 300 people were transported from the hospital to the nearby countryside. A mass gravesite discovered shortly thereafter at a farm near Ovcara was thought to contain the bodies of those who had been in the hospital. In 1995, at the request of the International Criminal Tribunal for the former Yugoslavia (ICTY), an international team of forensic investigators excavated the gravesite. Remains of 200 people, 198 men and 2 women, were exhumed.14 For each set of remains, morphometric traits were documented, as was location within the gravesite and proximity to any documents, clothing, or other belongings that might indicate identity.
Three sets of materials were sent to our laboratory for DNA sequencing: tooth and bone samples from remains, blood samples from surviving relatives of missing individuals, and blood samples from volunteers from the general population to serve as population controls. We received samples of tooth or bone from 62 individuals, 59 of which yielded adequate mtDNA for sequencing. The mtDNA sequences of 233 maternal relatives of missing family members and of population controls were determined using DNA extracted from blood. The mtDNA sequences of teeth and bone were compared to mtDNA sequences of maternal relatives. The frequency of each sequence was estimated from the population controls from the region and from our global database.
Of the 59 mtDNA sequences from remains, 46 matched the sequence of a relative searching for a missing person. In 21 cases the mtDNA sequence of the deceased individual matched only one family. In 26 cases the mtDNA of the deceased individual matched more than one family; that is, the sequence was public, at least locally. Additional evidence from the mass grave could be brought to bear on all these identifications, with focus restricted by mtDNA sequencing only to the families potentially related to each individual. The mtDNA sequences of 13 other individuals matched no survivors. These cases most likely represent individuals without living family members available to participate. Relatives of deceased were informed of results of the forensic analysis. Positive identification of the victims was included as evidence presented by the prosecutor of the International Criminal Tribunal of the former Yugoslavia at the Hague.14
The mtDNA sequences from the Vukovar project revealed interesting features of the genetics of maternal lineages of Bosnian, Croatian, and Serbian populations. The maternal lineages of these populations overlap. That is, individuals who self-identify as Croatian have mtDNA sequences found also within the Serbian and Bosnian populations. Similarly, individuals self-identifying as Serbian have mtDNA sequences found also in the Croatian and Bosnian populations, and so on. Despite their ethnic and religious divisions, there are no significant differences in frequencies of mtDNA variant sites among these groups.
Conclusions
We have described how our approach has been applied in Argentina and in Croatia. We have applied the same approach to human rights cases in El Salvador, Guatemala, Haiti, Rwanda, Mexico, Chile, Honduras, Ethiopia, Philippines and elsewhere in the Balkans. Human rights violators have used increasing levels of technology to perpetrate their crimes. Genomic tools provide an opportunity to use advances in biotechnology to combat these crimes worldwide.
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