The 2,049th Meeting of the Society

November 3, 1995

DNA Identification of the Last Romanovs

Thomas J. Parsons

Armed Forces Institution of Pathology

About the Lecture

DNA testing has played a principle role in solving one of the most dramatic and longstanding forensic mysteries of the century: the fate of Russian Tsar Nicholas II and his family. This work, performed at the Armed Forces DNA Identification Laboratory (AFDIL) and elsewhere, will be described, with emphasis on the use of mitochondrial DNA analysis of ancient remains. In addition to the remarkable and intricate human-interest aspects of the Romanov case, the lecture will highlight recent advances in molecular biological technology and genetic characterization that are the basis for determining personal identity from a wide range of biological samples. Much genetic information can be recovered even after great periods of time—years, decades, centuries, and beyond. This type of testing is regularly performed at the AFDIL to aid in the identification of U.S. service personnel from both present and past (Viet Nam, Korea, World War II) eras.

About the Speaker

Thomas J. Parsons received a Bachelors in physics from the University of Chicago in 1982 and a Ph.D. in biochemistry from the University of Washington in 1989. Subsequently, he focussed on the study of molecular evolution and genetic variation in many species, including birds, insects, and humans. He performed extended postdoctoral study at the Smithsonian Institution's Laboratory of Molecular Systematics, and more recently was a research faculty member at the University of Nebraska. In his current position at the Armed Forces Identification Laboratory, Armed Forces Institution of Pathology, he specializes in human mitochondrial DNA variation and ancient DNA analysis.


The President Mr. Ohlmacher called the 2049th meeting to order at 8:22 p.m. on November 3, 1995. The Recording Secretary read the minutes of the 2048th meeting and they were approved. The President then read a portion of the minutes of the 442nd meeting November 23, 1895. The President introduced Thomas J. Parsons, of the Armed Forces Institute of Pathology to discuss “DNA Identification of the Last Romanovs”. Mr. Parsons began by explaining some of the techniques and methodology of DNA identification. This technology is based on the development within the last fifteen years of the use of restriction endonucleases, enzymes that cleave nucleic acids at specific points depending on the sequences of bases, and of the polymerase chain reaction, or PCR, in which another enzyme accurately reproduces large numbers of copies of nucleic acids. This technology makes it possible to study variations in DNA when only very small amounts may be available or when it has been degraded because of age. Samples of ancient DNA as old as 30 million years have been successfully analyzed. It is the primary responsibility of the Armed Forces DNA Identification Laboratory (AFDIL) at the Armed Forces Institution of Pathology to attempt the personal identification of recovered remains of armed forces personnel. The AFDIL presently maintains more than 1 million blood samples of current and former active duty armed forces personnel who served within the last few years. For those individuals who may not have identified blood or tissue samples, identification can still be attempted by comparing the remains with samples obtained from surviving family members. There are two types of DNA in human cells, nuclear DNA that is inherited equally from both parents and mitochondrial DNA that is inherited almost exclusively from the mother. The mitochondrial DNA which exists as a small circular genome, is preferred for the identification of degraded skeletal remains for three reasons. There are many copies of it in each cell and high copy number improves the reliability and speed of the PCR method. The mitochondrial DNA, in particular a region of it called the D-loop, mutates comparatively rapidly providing sequences that can be used like an individual signature or fingerprint. And it is maternally inherited without recombining through many generations, so sensitive comparisons can be made with even distant relatives as long as they are matrilineally related. DNA identification technology has been used to solve one of the most dramatic and longstanding forensic mysteries of the century, the fate of Tsar Nicholas II and his family. Some information about the final hours of the Tsar and his family has been offered in the testimony of Yanov Yurovsky who says he led the group that carried out the executions on July 16, 1918, at the request of Bolshevic leaders. After the Tsar, his wife the Tsarina, his son the Tsarevitch, his 4 daughters, doctor and several servants were executed it was thought necessary to destroy the bodies to prevent them from becoming objects of veneration and encouragement to opposition forces. On the night after the executions, the bodies were placed in a mine which had been located only with a great deal of difficulty. When grenades failed to collapse the mine, the bodies were removed the next night. Two of the smaller bodies, the son and possibly the youngest daughter Anastasia, were burned. The other bodies were thrown in a pit, doused with sulfuric acid and buried. From records in its files the KGB was able to find the remains in 1979, but the location was not publicly disclosed until the collapse of the communist government. Last year the remains of at least 9 adult individuals were recovered along with remnants of cloth, leather, metal, jewelry and broken ceramic jars which had probably contained sulfuric acid. In order to identify these remains Pavel Ivanov of the Russian Academy of Sciences obtained DNA samples from the bones and provided them to the AFDIL. The next problem was to identify and obtain blood samples from living matrilineal relations. Fortunately, the pedigrees of the royal families of Europe are fairly well documented. The Tsarina Alexandra was the sister of the maternal grandmother of Prince Philip Mountbatten who provided a blood sample. This sample was used to identify the remains of the Tsarina and three of her daughters. Which three daughters could not, of course, be determined. The maternal grandmother of the Tsar, Princess Louise of Hesse-Cassel was also the mother of the maternal grandmother of the Duke of Fife who provided a blood sample. The remains which most closely matched the sample provided by the Duke of Fife, were slightly ambiguous because a rare anomaly called heteroplasmy, essentially simultaneous multiple markers at a particular genetic locus. In order to make the identification more certain, the body of Tsar Nicholas' brother the Grand Duke Georgi who had died in 1899 was exhumed in 1994, and tissue samples were taken from the same bones as from the recovered remains thought to be the Tsar. The sample from the Grand Duke exhibited the same rare heteroplasmy as found in the recovered remains; the likelihood ratio that the recovered remains were related to the brother of the Tsar was 300,000,000 to 1. These results were reported in “The Final Identification of Tsar Nicholas II”. Since the execution of the Tsar and his family, there have been people who claimed to be the Tsarevitch Alexei or the Grand Duchesses Marie or Anastasia. Rejecting as very highly improbable the survival of a young, hemophiliac boy from what was by all accounts an extremely brutal execution, the only claimant with some credibility was Anastasia, also known as Anna Anderson. This woman was thought by others to have been a particular Polish girl of non-royal heritage who had been injured in an munitions plant accident toward the end of World War I and briefly hospitalized before disappearing. When this lady who claimed to be Anastasia died in 1984 her remains had been cremated and it was thought that it might never be possible to establish or falsify her claim. However, an investigation found there were still in existence a lock of her hair and a biopsy sample that had been obtained in 1979. When these samples were analyzed they appeared to be unrelated to the recovered remains of the Tsarina and her daughters. They did match a sample provided by a Polish matrilineal relative of the Polish girl who had disappeared. Mr. Parsons kindly answered a number of questions from the audience. The President thanked the speaker on behalf of the Society, introduced two new members, announced the speaker for the next meeting, restated the parking policy, and adjourned the 2049th meeting at 9:24 p.m. Attendance: 55 Temperature: 4.0°C Weather: rain Respectfully submitted, John S. Garavelli Recording Secretary