PAT334982.indd Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Pathobiology 2012;79:228–238 DOI: 10.1159/000334982 Genetic Identification of Missing Persons: DNA Analysis of Human Remains and Compromised Samples M.J. Alvarez-Cubero a M. Saiz a L.J. Martinez-Gonzalez b J.C. Alvarez a A.J. Eisenberg c B. Budowle c J.A. Lorente a, b a Laboratory of Genetic Identification, Department of Legal Medicine and Toxicology, Medical Faculty, University of Granada, Granada , and b Pfizer-University of Granada-Junta de Andalucia, Center for Genomics and Oncological, and Biomedical Research Center, Armilla , Spain; c Institute of Applied Genetics, Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, Fort Worth, Tex. , USA analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is inte- gral to resolving a number of serious criminal and civil con- cerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still re- quired, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymor- phisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of de- composed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers. Copyright © 2012 S. Karger AG, Basel Introduction: Forensic Genetics and Challenging Samples Since the advent of forensic DNA analysis there have been two main objectives: (1) the identification of those who could be the source of biological evidence, which includes associations of individuals due to some alleged Key Words Bioancestry � Databases � DNA typing � Forensic genetics � Genetic identification � Missing persons Abstract Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typ- ing provides genetic data from a variety of materials and in- dividuals, and is applied to many important issues that con- front society. Part of the success of DNA typing is the gen- eration of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, hu- man remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database con- tains 10 million profiles). The scientific, governmental and private communities continue to work together to standard- ize genetic markers for more effective worldwide data shar- ing, to develop and validate robust DNA typing kits that con- tain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of Published online: June 21, 2012 Dr. M.J. Alvarez-Cubero Laboratory of Genetic Identification, Department of Legal Medicine and Toxicology Medical Faculty, University of Granada Avenida de Madrid 11, ES–18071 Granada (Spain) Tel. +34 958 249 950, E-Mail mjesusac   @   ugr.es © 2012 S. Karger AG, Basel 1015–2008/12/0795–0228$38.00/0 Accessible online at: www.karger.com/pat http://dx.doi.org/10.1159%2F000334982 Genetic Identification of Missing Persons Pathobiology 2012;79:228–238 229 kinship; and (2) to exclude individuals wrongly associ- ated with evidence. The generation of reliable genetic profiles from unknown and reference samples, system- atic and objective interpretation practices, and providing a statistical evaluation of the results are tantamount to a robust forensic DNA identification program. These cri- teria, seemingly obvious today, were envisioned 25 years ago by the forensic geneticists that developed this field. Moreover, the standards of practice used for forensic DNA typing are dramatically impacting in a positive way on the standards of criminalistics. Concepts and topics related to quality control and assurance, valida- tion, proficiency tests, documentation and statistical evaluation, for example, are being reconsidered by foren- sic scientists in other disciplines to improve quality and reliability of their areas of forensic science. A further dis- cussion on the direction of the forensic sciences can be found in the National Academy of Science Report ( Strengthening Forensic Science in the United States: A Path Forward [1] ). Because of the success of forensic genetics in the iden- tification of sources of biological evidence, developmen- tal and innovative progress continues with more expec- tations of assisting investigators. Future endeavors could include predicting the phenotype of an individual from a bloodstain or human remains, determining factors re- lated to cause of death, better capabilities to type severe- ly damaged biological evidence, typing fetal DNA from maternal circulating blood, and novel genetic markers for identity testing and bioancestry, for example. These amazing realistic potential applications were not too long ago the substance of science fiction. However, to- day efforts on the generation of large databases to develop investigative leads when there is no known suspect(s) and improving capabilities to extract genetic information from challenged samples are driving devel- opments in human DNA identity testing. These two ar- eas are of forensic, criminal relevance and directly im- pact on society, as well as historic interests in a number of situations. Large databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), from forensic evidence, human remains, and direct and family refer- ence samples of missing persons. There is a demand for more typed samples to be placed in these databases to help develop more investigative leads for crime solving. This need has motivated the community (government, academia and industry) to work collaboratively to devel- op and validate standard DNA typing kits that contain the reagents necessary to type core identity genetic mark- ers and the concomitant automation of a number of pro- cesses (extraction, quantitation, amplification and to some degree interpretation of the results) to facilitate typ- ing. The ability, for example, to type a reference blood or buccal sample without extracting the DNA away from interfering compounds would not have been imagined possible a few years ago and yet today such technology is readily available [2, 3] . The standard genetic markers used in essentially every forensic DNA typing laboratory in the world are autoso- mal short tandem repeat (STR) loci [4, 5] . The standard operating procedures employ a set of 10–17 STR loci, which provide a high level of diversity and resolution for identity testing [6–8] . Commercially available kits, such as the AmpFℓSTR � Identifiler � PCR amplification kit (Applied Biosystems, Foster City, Calif., USA) or the PowerPlex � 16 system (Promega, Madison, Wisc., USA), enable analysis with high-quality materials and forensic samples. These kits, and STR loci, have been used exten- sively for the identification of human remains as well as in kinship cases, such as paternity testing and family re- constructions. While these STRs apply to the majority of biological evidence analyses, there are situations where autosomal STRs cannot yield sufficient information. Ev- idence, such as mixtures with a large amount of female DNA and a small amount of male DNA, and kinship cas- es where the reference sample derives from a relative sep- arated several generations from the individual of inter- est require other markers, e.g. Y-STRs, X-chromosome- linked markers or mitochondrial DNA (mtDNA). These lineage-based systems provide additional power because of their unique biological qualities compared with auto- somal markers. Additionally, when performing familial searching, i.e. searching for relatives of the true source of the sample, these lineage markers are extremely useful for reducing the number of adventitious associations in can- didate lists. mtDNA sequencing is used to strengthen the genetic evidence when there are maternal relatives avail- able to serve as references [9] . mtDNA, inherited through the maternal line, has a special property that makes it particularly useful for samples that are severely degraded and/or of limited quality, such as human remains in miss- ing persons and mass disaster cases. There are hundreds to thousands of mtDNA molecules in a cell compared to only two copies of nuclear autosomal genetic markers [10] . Thus, when STR typing does not yield a result, there still is a good chance to obtain a result via mtDNA typing. In the following sections, the application of DNA analysis will be described in different areas where the common issue is the association of individuals via kinship analyses Alvarez-Cubero   /Saiz   /Martinez-Gonzalez   / Alvarez   /Eisenberg   /Budowle   /Lorente   Pathobiology 2012;79:228–238230 either for identifying human remains, combating human trafficking or historical studies. These approaches gener- ally share the process of indirect genetic comparisons to determine the identity of an individual and often the samples are compromised. The examples used are based on our own experience in developing and establishing missing persons’ databases [in Spain and in the US, the so-called FENIX and the Missing Persons Program at the University of North Texas (UNT) Center for Human Identification (CHI), respectively], missing children identification, which is embodied by the DNA-PROKIDS (Program for Kids Identification with DNA Systems) program, and attempts to identify persons of historical interest. Identification of Missing Persons Strategies for the Identification of Missing Persons The identification of human remains belonging to missing persons is one of the main challenges for forensic genetics. These identifications are a universal problem. When other human identification forensic techniques (e.g. dactyloscopy, anthropology, odontology and medi- co-legal examinations) provide limited information, or to support further or refute potential associations, DNA typing can be extremely valuable. DNA, in theory, can be recovered essentially from any tissue (e.g. soft, de- graded tissues, bones, teeth and hairs). In addition, be- cause close relatives on average share more genetic vari- ants than unrelated individuals, relatives can provide reference samples to effect identifications of unknown individuals or their remains or, at least, to develop poten- tial investigative leads for law enforcement. Lastly, DNA analysis can be a powerful exculpatory tool eliminating wrongly associated individuals, reducing candidates and redirecting resources to more viable avenues of investi- gation. The Spanish Phoenix Program (Programa FENIX) In November 1998, the Spanish Ministry of the Inte- rior decided to support an initiative from the University of Granada that was presented to the Guardia Civil to implement a National Program to attempt to identify ca- davers and bones from missing persons. The program was named ‘Phoenix Program’ (Programa Fénix, in Span- ish) based on its purpose with the name derived from classic Greek mythology. The Phoenix program contains two independent da- tabases (the questioned database , containing STR profiles and mtDNA sequences from bones, and the reference da- tabase , containing STR profiles and mtDNA sequences from relatives). The databases house genetic data that can be compared automatically to associate matching or re- lated profiles, such as those from unknown remains and reference samples from relatives [11, 12] . The general procedure is shown in figure 1 . Only per- sons signing a valid informed consent protocol are al- lowed to participate in the program and provide reference samples. People that have reported missing relatives are requested to contact the Phoenix Program by calling a toll-free telephone number. For those who voluntarily provide reference samples, two buccal swabs are ob- tained. A minimum of 2 and a maximum of 4 relatives (when available) can be sampled. All samples are bar cod- ed and subsequent genetic data are coded to maintain confidentiality and reduce the misuse of genetic data. For questioned database samples, typically 2–4 fragments of at least 25 g of compact bone and/or teeth from non-iden- tified cadavers and human remains are provided by trained specialists of the Guardia Civil. Autosomal STRs and mtDNA analyses are routinely performed. In cases requiring additional information, such as when a maternal reference sample is not available, Y-chromosome STRs can be included. STR typing is fa- cilitated due to the advent of multiplex commercial kits that provide the necessary reagents to type up to 15 STRs and a gender determination maker known as amelo- genin. These kits are the PowerPlex16 kit (Promega) and the Identifiler kit (Life Technologies-Applied Biosys- tems). Nowadays, there is an improvement in the Identi- filer kit (Identifiler Plus kit) and PowerPlex kit (Power- Plex 16 HS) which allows for better data recovery from degraded samples and a greater sensitivity of detection. Since these kits or variations of these kits are used world- wide, there is a high likelihood that any forensic or refer- ence sample that is typed by a forensic laboratory will contain common genetic markers. Therefore, genetic re- sults can be readily compared regardless of the country or laboratory that generates or maintains the data. For mtDNA analysis, DNA from 1 buccal swab from 2 maternally related individuals per case is extracted and sequenced for the 2 most informative regions of the mtD- NA genome. The regions are the hypervariable region 1 (HV1) and hypervariable region 2 (HV2) of the control region or d-loop of the mtDNA genome. To date, 1 3,700 families have contacted Phoenix, 862 have enrolled in the program and at least 319 unidentified remains have so far been identified and returned to their relatives. When mtDNA and/or STR associations are Genetic Identification of Missing Persons Pathobiology 2012;79:228–238 231 found, a second independent analysis is performed as part of the quality assurance process. Nationally and internationally compatible protocols leading to the identification of human remains or skele- tons ideally will require the use of databases that meet four basic prerequisites, as follows: (1) Analyses have to be based on standard operating protocols and universally accepted genetic markers. (2) Results must be reliable (laboratories and tech- niques are subjected to quality assurance and quality control programs). (3) The technology should be amenable to automation, to facilitate the typing of the anticipated large volume of samples and to allow intra- and international searches and comparisons. (4) The data provide little or no personal or confiden- tial information about the individual(s). Proper use of the database according to national laws, dissociation of data, restricted access, informed consent from voluntary do- nors and court orders to handle human remains are among some of the requirements of Spanish database management. The Phoenix program is using DNA to develop asso- ciations between relatives of missing persons and uniden- tified cadavers or human remains of previously unsolved cases. Once an association is found, anthropologists, odontologists, specialists in forensic medicine and law enforcement officers work together to establish a final, positive identification and prepare a report for the Court (http://www.guardiacivil.es/prensa/actividades/fenix/ presentacion.jsp). The US Missing Persons Program In the US, the Federal Bureau of Investigation (FBI) facilitates similar databases of missing persons and un- identified human remains, the NMPDD (National Miss- ing Person DNA Database) Program. The database con- tains three indexes in which DNA profiles can be entered: biological relatives of missing persons , unidentified human remains and missing persons . There are only a handful of qualified laboratories in the US that have full capabilities to analyze missing person cases. The capabilities include typing the full battery of genetic markers, i.e. autosomal STRs, Y-chromosome STRs and mtDNA (http://www. f bi.gov/about-us/lab/dna-nuclear). Nowadays, in the US, a total of 4,285 families have re- ported missing relatives whereas only 3,461 unidentified human remains have been found. The largest program of missing person identification in the US, however, is in the State of Texas. The Texas Questioned samples (non-identified corpses and human remains) Reference samples (relatives: voluntary) (1) Judicial petition (2) Exhumation or autopsy samples (3) Laboratory: genetic identification (1) Contact FENIX Program (900150759) (2) Inform the relatives (3) Collaboration of the relatives: • Informed consent • Voluntarily (4) Collect saliva samples (5) Laboratory: genetic identification Reference database Questioned database Comparison Matching nuclear DNA and/or mtDNA and/or Ychr DNA (1) Anthropologic and odontologic data (2) Other forensic data of interest (3) Police reports Final report - Court - Relatives Data eliminated from the database Fig. 1. Identification of corpses and human remains in the Phoenix program. Ychr = Y chromosome. Alvarez-Cubero   /Saiz   /Martinez-Gonzalez   / Alvarez   /Eisenberg   /Budowle   /Lorente   Pathobiology 2012;79:228–238232 Missing Persons DNA Database was established in 2001 at the UNTHSC. UNTHSC in collaboration with law en- forcement offers families with missing loved ones the op- portunity to submit reference samples for DNA testing. The laboratory is one of only a few facilities that inte- grates nuclear DNA and mtDNA for analyses. Once DNA profiles are obtained, they are directly entered into the FBI Combined DNA Index System plus Mito (CODIS+ mito) database. The database began accepting samples from Texas law enforcement agencies in March 2003. Texas was the first state in the country with a missing person DNA database capable of analyzing both mitochondrial and STR sys- tems and is the first state to participate in the federal da- tabase for missing persons (the Federal Bureau of Inves- tigation’s CODIS). The database provides a very power- ful tool for investigators trying to locate missing persons or identify remains by allowing federal, state and local crime laboratories to electronically exchange and com- pare DNA profiles. The DNA analysis provided by the Texas missing person DNA database is at no charge to law enforcement agencies or families with missing mem- bers. The UNTHSC in Fort Worth, Tex., USA, is home of the UNTCHI DNA laboratory established in 2001. In collaboration with law enforcement agencies, medical examiners and coroners throughout the nation, the UNTCHI has established one of the largest missing person programs in existence. The program offers the families of missing loved ones the opportunity to submit reference samples for DNA testing. The laboratory is one of only a few facilities that integrates the analysis of nu- clear DNA and mtDNA. UNTCHI incorporates the use of the FBI CODIS+mito database to locate missing per- sons and identify human remains. The database operates at a local (LDIS), state (SDIS) and national level (NDIS). Texas was the first state in the country with a missing person DNA database. UNTCHI services are performed at no charge and have expanded to a national level. To date, UNTCHI has completed the analysis of 7,792 fam- ily reference samples, 3,461 unidentified human remains and 182 direct reference samples. Once completed, these profiles must meet eligibility requirements in order to be uploaded to the next level of CODIS. Of the eligible com- pleted samples, a total of 5,300 family reference samples, 2,450 unidentified human remains and 140 direct refer- ence samples have been entered into the local CODIS da- tabase. Currently, UNTCHI has made 700 associations through the use of CODIS. Missing Children Identification Children Trafficking and Exploitation Children, the most innocent individuals of society who should be protected to the best of our abilities, can be subjected to many abuses. One of those abuses is hu- man trafficking, an apparently lucrative criminal activi- ty. According to UNICEF, ‘an estimated 300 million chil- dren worldwide are subjected to violence, exploitation and abuse including the worst forms of child labor in communities, schools and institutions; during armed conf lict; and to harmful practices such as female genital mutilation/cutting and child marriage’. Figures from the United States only begin to demonstrate the magnitude of the missing children problem within a country. Ap- proximately 800,000 children are reported missing each year. Of these, approximately 360,000 are runaways, 340,000 are classified as ‘missing with benign explana- tion’, and about 100,000 are abducted either by family members or other known individuals or are lost and/or injured [13] . While these figures are disturbing, they re- late to mostly domestic situations and do not represent the greater international problem where children are il- legally sold for often malevolent purposes. We must ensure that children are treated humanely and in keeping with national and international stan- dards. In an effort to combat human trafficking and es- pecially to protect children (and women), the United Na- tions (UN) launched the UN-Global Initiative to Fight Human Trafficking (UN.GIFT) in March 2007 (http:// www.unodc.org/). Such initiatives demonstrate that gov- ernments recognize the problem of human trafficking is substantial and steps must be taken to eradicate this vic- timization of children. However, there are several obsta- cles to combating human trafficking. International agreements and policies must be in place to track, iden- tify, communicate and share data, as well as interdict per- petrators and help victims. Worldwide political and legal coordination plays a crucial role, but the legal and social difficulties that exist for access and disclosure among countries substantially slow the progress of implement- ing a comprehensive and effective counter-trafficking system. Scientists have the technology tools to assist in the identification of missing persons and yet may not be able to apply them in the international endeavor to fight hu- man trafficking. The use of different genetic markers and/or incompatible software would obstruct this effort, which is especially troubling when children are victims. Indeed, genetic testing capabilities for identification are Genetic Identification of Missing Persons Pathobiology 2012;79:228–238 233 readily available and implementation may seem trivial compared with overcoming legal infrastructure barriers and addressing privacy concerns. Because legal infra- structures will be slow to develop it is important that the construction of the technical infrastructure should pro- ceed in preparation for the implementation of an appro- priate international legal framework. This will facilitate efforts in thwarting human trafficking, once the policies for sharing data are addressed adequately. One technical area sufficiently developed for implementation is the use of the molecular biology analytical tools (i.e. DNA test- ing) and databases for the identification of missing chil- dren. The DNA-PROKIDS Program: DNA to Fight Crime on Children Because of the importance of the children trafficking problem and based on our experience with the Phoenix Program [14] and those at the UNTHSC and the UNTH- SC Center for Human Identification (UNTHSCCHI), we have launched DNA-PROKIDS located at the University of Granada and in collaboration with UNTHSCCHI. The Program is an international effort to help identify missing children, provide support to their relatives and to contrib- ute to efforts against human trafficking. This non-prof- it program is supported by the Spanish Government, the Andalusian Government and donations from pri- vate companies and foundations (BBVA, Banco San- tander, CajaGranada-BMN and Life Technologies). DNA- PROKIDS is composed of three tiers. The first tier is at the National level with two genetic databases or indices per country. One index is for DNA profiles (and meta data) obtained from children who, after proper investigation, are found in an illegal situation (e.g. not living with the natural family due to abduction, kidnapping, illegal adop- tion or other criminal situations). The other index com- prises DNA profiles (and meta data) voluntarily provided by relatives (parents, sibs and other meaningful family members, and whenever possible obtaining mothers) or from personal items of reported missing children ( fig. 2 ). The DNA profiles in these two indices will be compared routinely to assist in identifying missing children initially within their own countries. DNA-PROKIDS first tier pi- lot programs are in effect in Guatemala and Mexico. Ad- ditional efforts are currently underway in Brazil and Chi- na. It would be highly desirable to coordinate these ef- forts. Without coordination it is feasible and somewhat tragic that systems may be developed which are incapable of data exchange because of the use of non-overlapping genetic markers or incompatible software, or both. The power of identity testing and database searching will be most effective if DNA analysis is performed in ev- ery single case of a child being given to adoption. Fur- thermore, when possible, the mother (or other available biological relatives) should also be tested to confirm her relationship to the child and therefore right to relinquish the child. Implementation of this act alone would reduce crimes related to children, where the children are given to adoption not by their biological families, but after kid- napping or abduction. The second tier is at an International level. This will allow the development of the infrastructure required to share data among countries through various existing ap- proved links and networks as well as through the estab- lishment of new, specific links. International cooperation requires decisions on many issues, including a common set of DNA markers to enable genetic data sharing; the meta data that should be collected; the information that can be shared; legal and privacy issues that need to be met to share data, and the development of sufficient sustain- able financial support to establish such programs in var- ious countries. The third tier is focused on data generation. There will be a universal automatic, mandatory inclusion (with sig- natory countries) in the database of (1) any reported child found out of his/her family (without legitimate reasons); (2) children who are going to be adopted (and before any adoption can be made it will be necessary to confirm that the child has not been reported as missing anywhere in the world), and (3) the immediate inclusion of voluntary relatives of missing children. DNA-PROKIDS makes use of the same robust DNA typing methods used for forensic casework and those de- scribed above for missing person identification. These markers are extensively validated, and substantial data exist that support their utility. In addition to STRs and mtDNA, single nucleotide polymorphisms (SNPs) are particularly suited for a program like DNA-PROKIDS [15–18] . SNPs are genetic variants that are the result of substitutions or insertions/deletions at one or a few bases in the genome. They occur at about 1 SNP/1,000 bp in the human genome and account for approximately 85% of human genomic variation. Millions of SNPs have been identified and a subset of these is suitable for identity test- ing. They would help to increase the probability or likeli- hood ratio in cases of positive associations, and also to overcome problems related to mutation that could occur more so with STRs and mtDNA. The application and usefulness of DNA identity test- ing are already well documented. To date, DNA- Alvarez-Cubero   /Saiz   /Martinez-Gonzalez   / Alvarez   /Eisenberg   /Budowle   /Lorente   Pathobiology 2012;79:228–238234 PROKIDS participating countries have analyzed over 2,500 cases (basically from Mexico, Guatemala, El Salva- dor, Paraguay, Peru, Bolivia in Latin America, and the Philippines, Thailand, Indonesia and India in Asia). DNA analyses first and subsequent application of ac- companying meta data have already helped to identify 1 330 missing children, who have been returned to their families. If not for this intervention, it is likely these chil- dren would have been given or sold into illegal adop- tions, would still be under exploitation or would have died without identification. Additionally, their respec- tive families would still be suffering the loss of their chil- dren. Beyond the identification of these children and re- turning them to their families, the database could play a deterrent role. Efforts that increase the size of the data- base and facilitate communication among countries, such as is encountered with various criminal investiga- tions through INTERPOL, may make criminals more reluctant to commit these heinous crimes on children (trafficking, exploitation and illegal adoptions) because at least authorities will more likely be able to identify and apprehend perpetrators. If these programs were enacted, the ability to immedi- ately identify reported missing children would not only permit returning them to their families, but also would begin to compromise criminal network operations. More operational data and updated information can be found at www.dna-prokids.org . Historical Cases and DNA Identification There have been a number of historical individuals whose identity of putative remains have been confirmed or questioned by DNA typing. These include Tsar Nicho- las, St. Birgitta, Napoleon, or African-American descen- dants of Thomas Jefferson’s line, for example. Typically, mtDNA and Y-STRs, and at times autosomal STRs, play important roles in historical analyses. We have had the opportunity to participate in a num- ber of historical identifications in Spain. In 1994, a set of remains thought to be those of Queen Blanca I of Navar- ra were found in a church in the village of Santa María de Nieva, province of Segovia, Spain, where it is known that she died on April 3rd, 1441. The anthropological analysis Fig. 2. Extraction kit of DNA-PROKIDS. Genetic Identification of Missing Persons Pathobiology 2012;79:228–238 235 did tentatively conclude that the remains could be the ones of Queen Blanca, but because of the data and chang- es to different graves, DNA analysis was requested. Ac- cording to historians and experts, the Regional Govern- ment of Navarra (northern Spain) decided that the refer- ence for DNA analysis should be the Prince Carlos of Viana, the son of Queen Blanca. It is also known that the Prince of Viana was buried at the Monastery of Poblet, in the province of Tarragona, Spain. When we proceeded with the exhumation of Prince Carlos, the remains inside the coffin were found to belong to at least 3 different individuals based on skeletal mor- phology. This observation had a historical explanation. Poblet’s monastery had been assaulted and its tombs des- ecrated a number of times during the XIXth century. mtDNA analysis performed on the putative remains of Queen Blanca and his son Prince Carlos [around 1994 and 1995 – at the University of Granada and at the Pennsylva- nia State University (Dr. Mark Stoneking and Dr. Anne Stone) – did confirm the initial findings. The coffin of the Prince of Viana contained at least 3 persons. Furthermore, none of the mtDNA sequences obtained from the remains that could have been the ones from the Prince matched the sequence generated from the remains who are thought to be the ones of Queen Blanca [unpubl. results]. Therefore, DNA analysis could not resolve the authen- ticity of the remains of the Queen. They did support, however, that the remains in Poblet are not likely those of Prince Carlos. Studies are on the way to find an appropri- ate reference sample. The process is a very difficult one because most of the reference samples belong to kings, queens and other members of royal families, and permis- sion for sample access is often hard to obtain. The second project where our teams have been in- volved is in the identification of the remains of Christo- pher Columbus, who died in 1506 in Valladolid (north- western Spain). His bones reportedly were moved to Se- ville (Spain) in 1509 and then from Seville to Santo Domingo (Dominican Republic) in 1544; in 1795 from Santo Domingo to Havana (Cuba), and finally, in 1898, the remains of Columbus (or what it was thought to be the remains of Columbus) were sent back to Spain, and buried at the Cathedral of Seville. Because the remains were subjected to 4 different transfers and because of some historical doubt regarding the authenticity of the remains buried in Seville, a group of historians led by Mr. Marcial Castro initiated a project (still under development) to first try to determine if the remains in Seville are the ones of Columbus. Second, he sought to undertake a study on the Y chromosome to de- termine objectively the origins of Columbus. It is widely accepted that Columbus was an Italian sailor, but some historians propose that he was of Spanish or Portuguese nationality. On June 3rd, 2003, the purported remains of Christo- pher Columbus and those from his son Hernando Co- lumbus (Cristóbal Colón and Hernando Colon in Span- ish, respectively) were exhumed in the Cathedral of Se- ville in Spain, and moved to the Department of Legal Medicine of the University of Granada. Prior to receiving the alleged Columbus bones (September 2002), the re- mains of the brother of Christopher Columbus, Diego Columbus, had been exhumed from its grave in Santi- ponce, a village close to Seville ( fig. 3 ). DNA analysis was performed in parallel by teams of the Universities of Barcelona, Santiago and Granada (Spain), Univeristy of Tor Vergata (Italy) and the Max Planck Institute for Evolutionary Anthropology. The consensus results showed a match in the sequence of mtDNA analysis of the remains of Diego Columbus with the ones thought to be of his brother, Christopher Colum- bus. Although the anthropological analysis was very lim- ited because of the size and degradation of the remains, the analysis also supported that the remains could be those of Columbus. Nevertheless, it must be mentioned that the set of re- mains buried in Seville and identified as the ones from Columbus are just a part, probably no more than 30–40% of the whole remains that could arise from 1 person. It is therefore logical to consider that the remains claimed to be that of Christopher Columbus and buried in Santo Do- mingo, Dominican Republic, could also be from the Ad- miral and conqueror. DNA analysis should be authorized by Dominican authorities and is pending on the samples from the Dominican Republic. Regarding the origins of Columbus, we have per- formed a study on Y chromosomes from people named ‘Columbus’ in Northern Italy (the regions of Liguria and Lombardy), and people named ‘Colom’ in the regions of Catalunya, Baleares and Valencia (Spain) and also some ‘Colom’ living in the Mediterranean French coast. The conclusion of this study [19] shows that the Y chromo- some from Italian Colombo men was more diverse than the Iberian Colom ones. In Italy, the Colombo surname arose many different times. Genetic and lineage diversity were greater in Lombardy (probably because the name used to be given to orphans and foundlings in Milan) and less pronounced in Liguria and Piedmont. This suggests that Columbus may be of Catalan descent. Alvarez-Cubero   /Saiz   /Martinez-Gonzalez   / Alvarez   /Eisenberg   /Budowle   /Lorente   Pathobiology 2012;79:228–238236 Additionally, this study shows that Colombo and Co- lom are two distinct surnames with no clear genealogical connection with local origins in Italy and Spain. How- ever, Colombo is more frequent in Italy (where it is actu- ally the most frequent surname in Lombardy) than Co- lom in Spain, and thus the number of Italian samples studied might not be equally representative. Since neither the Y-chromosome DNA of the remains of Christopher Columbus nor of his son Hernando has yet been ana- lyzed, no conclusions regarding the origins of Columbus have been proffered by us. In principle, other sets of markers can be used to at- tribute an individual to a population, and, given the vast number of markers, autosomal SNP arrays could be used. But the close similarity of the Catalan and North Italian general populations, as well as the poor suitability of this type of marker for ancient samples should be taken into consideration. For this reason, assessing and assigning the Columbus origin will be complex. Future Needs and Developments in Forensic DNA Analysis Human remains are exposed to a myriad of environ- mental challenges, as occurs with all forensic biological evidence. The result is that samples can be limited in quantity, degraded and contain contaminants that affect the ability to type DNA samples. The often compromised samples that are encountered in missing person identifi- cation are bones, teeth and hair, as well as poorly pre- served corpses, such as those found in water, buried or burned. It is very challenging, if not impossible, to extract genetic information from severely compromised DNA samples. To expand DNA typing capabilities on such challeng- ing samples, efforts are underway to extract more DNA from these materials, repair damaged DNA to generate more viable templates, improve PCR conditions to over- come troubling stochastic effects and develop alternate protocols for enhancing the sensitivity of detection. Un- derstanding the mechanism of degradation processes can provide insight into avenues for repairing DNA. Current- ly, attempts to repair DNA have come primarily from the ancient DNA arena where there is a greater chance that the bases in the DNA have been chemically altered. Ad- ditionally, New England Biolabs has produced a kit for repairing DNA that is being evaluated by our laboratories [20] . As mentioned above, the standard operating pro- cedures employ a set of STRs, as well as Y STRs and mtDNA. mtDNA typing is invaluable to missing person identifications, but it is a laborious, costly and time-con- suming process. New technology exploiting electrospray Fig. 3. Bone remains of Christopher Co- lombus relatives. Genetic Identification of Missing Persons Pathobiology 2012;79:228–238 237 ionization mass spectrometry with the PLEX-ID system (Abbott) enables automated, high-throughput mtDNA analysis [21] . A power of discrimination, approaching that of Sanger’s sequencing, is achieved but with reduced costs. In addition, some of the vagaries of mtDNA se- quencing, such as the inability to analyze heteroplasmic regions and mixtures, are overcome with the PLEX-ID [21] . While the autosomal and lineage markers provide a high level of diversity and resolution for identity testing, severely compromised samples may not contain suffi- ciently long enough template molecules to yield results with the current format kits. Therefore, alternate genetic markers that may be more applicable to much degraded DNA are being sought. SNP variation is restricted to a small site of the genome. Thus, the amplicon generated that captures the SNP can be smaller in size than those generated for STRs. Because it is feasible to reduce the amplicon size for SNP typing to only 60–80 bp in length [22] , much more degraded DNA samples can be typed than with the mainstay STRs. A single SNP is not as in- formative as a single STR locus; most SNPs are bi-allelic. However, technology exists to multiplex a large number of SNPs to obtain identity testing power equivalent to that afforded by using multiplex STR kits. The SNPs can be divided into five classes [23–25] based on their application: (1) Identity-Testing SNPs , which are those that have the desired features of high heterozygosity and low population heterogeneity [26, 27] ; (2) Lineage-Informative SNPs , which are sets of tight- ly linked SNPs that function as haplotype markers, or as pseudo-STRs, and are particularly useful for kinship analyses [28] ; (3) Ancestry-Informative SNPs , which are SNPs that differ substantially in frequency in population groups and can be used to reconstruct an individual’s biogeographical ancestry; (4) Phenotype-Informative SNPs , which are SNPs that can be used to directly recon- struct an individual’s phenotypic characteristics, such as skin, hair and eye pigmentation, height and facial fea- tures [29] , and (5) Pharmacogenetic SNPs , which are SNPs that can be used to determine cause and manner of death based on an individual’s genetic predisposition to triggering risk events. The forensic DNA field is inves- tigating these markers, and we fully expect SNPs to be an important part of the forensic DNA repertoire in the not too distant future. Lastly, the capabilities of next-generation (or actually better termed current-generation) sequencing have im- proved and costs have dropped dramatically. The ex- amination of large parts of the genome will make inter- pretation of difficult mixtures easier and will facilitate research to identify the next generation of forensic markers. Already whole genome sequencing has been shown to be effective for microbial forensic investiga- tions [30] . However, with these more-resolving and greater-depth tools, there is a concomitant gathering of private or personal data. Serious thought should be giv- en to the degree of information that should be typed and/or disclosed. 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