Peru is a country with a rich history and culture. It was home to one of the oldest civilizations in the world, the Norte Chico, and to the largest pre-Columbian civilization, the Inca Empire. Its rich land attracted conquerors, colonists, and immigrants from every continent over its five centuries of recorded history. As a nation, Peru has had many periods of political stability and economic growth. However, between 1980 and 2000, the country was torn by civil war. Around 15,000 people disappeared during that period of political upheaval, and their remains are still being uncovered in nameless graves around the country. It is with these two aspects of Peruvian reality that our guest speaker is concerned: the genetics of its population and its unidentified dead.
Gian Carlo Iannacone de la Flor is the Head of the Laboratory for Molecular Biology and Genetics of the Institute of Legal Medicine and Forensics in the Ministerio Público of Peru. His major research area is Populations Genetics, with a focus on the genetics of cancer, forensic genetics and animal genetics. He also teaches courses in forensic, evolutionary and medical genetics. Thanks to training in the labs of the International Commission on Missing Persons (ICMP), he deepened his knowledge of the extraction of DNA in difficult cases like the ones he and his team have to deal with in Peru. He has always been at the forefront of those focusing on the application of genetics to discover connections between people and identity.
What have been your main areas of research?
In the field of forensic genetics, I was part of the group that designed and implemented the DNA Laboratory in the Institute of Legal Medicine in Lima, Peru in 2002. In the case of cancer genetics, I have conducted research on the relationship between susceptibility to lung cancer formation and the presence of phase II genes. I am also investigating the potential correlation between genetic polymorphism and patient response to chemotherapy. In the field of animal genetics, I have done work with paternity, genealogy reconstruction, and population structure.
What are your current main research interests in population and forensic genetics?
I am currently interested in a broad range of topics. In the field of forensic genetics, I am looking at X-chromosome STR markers and European STR markers within the Peruvian population structure. In more practical forensic projects, I am interested in the automation of DNA extraction from skeletal remains without the use of phenol-chloroform. The relationship between causes of death and phase I and II genes, and CYP2D6 polymorphism as a marker of cause of death are other major areas of interest.
You mentioned the automation of DNA extraction from skeletal remains. Is this in connection to the missing persons project?
Yes, it is. We have a very large volume of work to do. Between 1980 and 2000, during the civil war, around 15,000 people disappeared in Peru. Many were buried in nameless graves. Some of their remains were left to lie in acidic soils, which makes it particularly difficult to find and extract genetic material.
In 2009, we began to improve the processes for obtaining DNA from skeletal remains and making genetic profiles of the victims. We improved the cleaning and grinding of the skeletal remains, and the system of DNA extraction. During a trip to the ICMP facilities in Bosnia we saw how they deal with some of their purification issues. Afterward, we eliminated the use of organic purification, so now we do the purification with Amicon and with the QIAGEN Investigator Kits. The Investigator purification kit is processed using QIAcube.
Is your forensic research focused entirely on missing persons?
No, we also handle criminal cases. In terms of the use of DNA as evidence in court, we have several projects, including one to determine the origin of the sample donor through a genetic population study in some parts of Peru, applying a Bayesian system to determine the most probable origin of that individual.
What is your main research project in terms of population genetics?
We have a project to establish a genetic database for Peru, looking at the genetic structure of the multi-ethnic Peruvian population. We are using autosomal STRs to determine the population substructure and migration for the peopling of America and Peru. Currently, we have 10 sites sampled, which is approximately 5,000 samples collected. We are working on two populations of this group of samples. One is the Ayacucho people, where we find significant sub-structuring, and the other is the Puno people, where we want to determine differences among the north and south populations in Puno. These populations will be analyzed with X markers and European STR markers using QIAGEN kits like Investigator Argus X-12 and ESSplex SE.
Does this database also serve a purpose for your missing persons work?
Naturally, we will be using it for its broadest application. The other objective of this is to have the data in the CODIS database for forensic cases, and DNA•VIEW for missing persons. I saw the software used in the ICMP facilities, and my conclusion is that it could be a good system for obtaining identification probability. This will be financed by one or more international organizations, probably the IOM, the World Bank, or the Red Cross.
Could you tell us a little about your laboratory?
Our main focus is on human identification. Our purpose is to determine paternity, obtain the identity of missing persons, and identifying culprits in criminal acts.
We have a central laboratory in Lima, where we work on criminal and paternity cases. Our other three laboratories are in the provinces, one in the north and one on the south coast, both for paternity cases, and one in the mountainous area south east of Lima. There, we will perform only work on DNA extraction from the skeletal remains of missing persons who died by violence in our country between 1980 and 2000.
We have a great deal of automation, and its implementation was supported by the Head of the Institute of Legal Medicine, Dr. Gino Davila Herrera. He is the supervisor of our Institute’s development.
What kind of sample numbers do you process?
In our laboratory, we work about 100 cases per month, but that is only our paternity and criminal caseload. Our total forensic sample load is 1,500 samples and 1,000 reference samples. In terms of the missing persons cases, there are approximately 24,000 reference samples, and a total of 8,000 skeletal remains to be identified. The sample load comes to around 1,700 samples per month. So we have a total monthly processing load of around 3,200 samples.
What are the key steps in your workflow?
Sample collection and transport are key areas. If these are done correctly, the extraction process has the chance to be successful.
Our transport process uses swabs and FTA cards. Keeping the samples dry and ensuring chain of custody are also very important, and we have appropriate envelopes for this.
To improve the genetic profiles in forensic cases, it is necessary to have a good system of DNA purification and recovery to ensure the removal of the inhibitors and obtain more DNA.
In the case of degraded DNA, I recommend using miniSTRs to ensure a complete profile. Using bi-allelic markers like DIPs or SNPs may be an additional option for achieving higher discrimination power. It is important to note that the use of real-time PCR is essential between the extraction phase and the amplification of STR phase, which is used to make decisions in the processing of the samples. Also it would be useful to have a commercial kit to measure mitochondrial DNA, which is important in cases where there is very little nuclear DNA as a consequence of degradation.
What QIAGEN products do you use in your workflow?
We use the QIAcube for DNA purification from bone samples, and QIAgility to prep PCR for quantification and STR amplification. As I mentioned earlier, we use the QIAGEN Investigator Kits as well.
What are your plans for the future?
In the future, we hope to have a network of laboratories working with robotic systems for DNA extraction and quantification and the amplification of STRs. As I mentioned, I want to see all of the information generated entered in the CODIS system for forensic cases and another set of genetic databases of missing persons.
Ultimately, we want to have a precise molecular system for determining the cause of death in genetic terms.