By Amanda Gardner
WEDNESDAY, March 11 (HealthDay News) -- An enduring mystery has been laid to rest with the DNA identification of the bodies of two children of the last Tsar of Russia.
The bones of the siblings, Tsarevich Alexei and a sister, were discovered in a grave outside Yekaterinburg in 2007. The remains of their father, Tsar Nicholas II, the Tsarina Alexandra and their three other daughters were found in 1991 about 70 meters away and were subsequently identified.
"The DNA evidence is strong, but if you if you look at the entire evidence, it's very convincing that this was, in fact, the Romanovs," said Michael Coble, lead author of a study published in the March 11 online issue of PLoS One and research section chief of the Armed Forces DNA Identification Laboratory.
Other evidence included three silver amalgam fillings on the crowns of two molars which undoubtedly belonged to an aristocrat.
The U.S. researchers worked with the Prosecutor's Office of the Russian Federation, which was treating the case like an unsolved homicide, Coble said.
"In many ways, it's not unlike a lot of missing person and unidentified cases where we have no bodies," said study co-author Anthony B. Falsetti, a forensic anthropologist with the University of Florida in Gainesville.
Over the years, skeptics have argued that the remains had not definitively been identified as those of the ill-fated Romanovs.
The Tsar, his entire family and four staff members were killed by a Bolshevik firing squad early on the morning of July 17, 1918.
Apparently, the executioners had attempted to destroy the bodies of Alexei and his sister (either Maria or Anastasia).
"The historical record is that when the Bolsheviks were disposing of the bodies, they took two of the remains and tried to cremate them to try to get rid of all of the evidence. They did a test [on the recently discovered two bodies] to see how it worked, and it didn't work that well. It took them all night," said Coble. "It takes a very high temperature to cremate a body, and when you're out in the woods, you don't necessarily get that kind of heat. The executioner had actually brought in an expert on cremation, but apparently, the guy broke his leg. It was a ridiculous sequence of events."
The end result was that these two bodies were buried in one grave and the rest of the bodies in another, a fact that had fueled speculation that two of the children had managed to elude the bullets and escape.
Based on DNA technology available in the 1990s, the first five bodies were positively identified as the Tsar, the Tsarina and three of their children.
The second set yielded 44 bone fragments and teeth which were subjected to three types of genetic testing: mitochondrial DNA, autosomal STR and Y-STR testing.
The mitochondrial tests (mitochondria are passed through the mother) confirmed that the bodies were children of the Tsarina. Confirmation was done with a living maternal relative, HRH Prince Philip, Duke of Edinburgh.
The autosomal STR test was basically a paternity test, Coble said, revealing that the newer remains are 4.3 trillion times more likely to be related to the Tsar and Tsarina than two random individuals. Skeletal remains from the three daughters found in the first grave also matched up.
"It's as strong as evidence you're going to find as far as nuclear DNA goes," Coble said.
The Y-STR testing, done only on the remains of Alexei, matched the STR profile of the Tsar and also a living relative, Prince Andrew Romanov, a cousin of the Tsar.
The STR and Y-STR findings were confirmed by testing blood on a shirt that Nicholas had worn on a trip to Japan in April 1891, when he was attacked with a saber. The shirt had been stored at the Hermitage Museum.
The results were confirmed, reconfirmed and confirmed again by independent labs.
One mystery remains however: the identity of the daughter found in the later grave. Russian forensic anthropologists have insisted and continue to insist that it is Maria. American experts assert it is Anastasia.
"For about 80 years, no one knew the real truth of where the bodies were taken, and you always had the romantic mythology of Anastasia, the youngest daughter, who was beautiful and adored and able to survive, because she charmed her captors into letting her go," said Coble. "Since 1918, over 200 different people have claimed to be one of the children. This truly now ends the case. You can no longer say, 'You never found the two children.' "
Visit romanov-memorial.com for more on the last Tsar of Russia.
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