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DNA Tests Confirm Remains of Russian Royals Killed During Revolution

NEW YORK (GenomeWeb News) – DNA evidence has verified that remains found in two graves near the Russian city of Yekaterinburg belonged to the Romanovs, Russia's last royal family. The research, scheduled to appear online this week in the Proceedings of the National Academy of Sciences, confirm that Czar Nicholas II and his entire nuclear family were killed during the Russian Civil War.

Researchers from the Russian Academies of Science and Medical Science, the University of Massachusetts, the Lomonosov Moscow State University, and the Canadian company Molecular World used DNA forensics to identify human remains found at two grave sites discovered more than 15 years apart. By isolating, sequencing, and piecing together mitochondrial and nuclear DNA from bone shards at each site, the team could compare the DNA with the family's living relatives and other sources.

Using this approach, the team confirmed that Nicholas II, his wife Alexandra, and three of their daughters were buried in one grave, discovered in 1991, while Prince Alexei and one of his sisters were buried in another, not found until 2007.

"The results of our studies provide unequivocal evidence that the remains of Nicholas II and his entire family, including all 5 children, have been identified," lead author Evgeny Rogaev, a University of Massachusetts neuroscience and genetics researcher who is also affiliated with the Russian Academies of Science and Medical Science, and his colleagues wrote.

The Russian Federation Prosecutor's Office recruited Rogaev to help out with the Romanov investigation, and he was officially assigned this case in November 2007. He and his colleagues reported their findings to Russian authorities last spring but the new paper represents the first publication of their genetic analyses.

Emperor Nicholas II, Empress Alexandra, their five children, three servants, and a court physician were killed by a Bolshevik firing squad in Yekaterinberg in July 1918, following Nicholas' abdication the previous year.

Although DNA studies supported the idea that remains found in a mass grave in Russia's Ural region in 1991 belonged to Nicholas II, Alexandra, and three of their children, initial genetic analyses generated some debate. Even so, the remains from that grave were reburied in the imperial resting place in St. Petersburg.

But the bodies of two children — Prince Alexei and one of his sisters — were missing from the 1991 grave, fueling previous speculation that one or more Romanov children might have escaped execution. "Legends persisted for a century that Alexei and Anastasia, the two youngest children of the Romanov family, survived those turbulent times," Rogaev and his co-workers wrote.

Then, in July 2007, anthropologists found more remains near Yekaterinburg — identified as a boy between the ages of 10 and 14 years and a woman aged 18 to 23 years.

In an effort to determine whether the new remains belonged to the two missing Romanov children and confirm the identity of all seven individuals, Rogaev and his colleagues isolated mitochondrial and nuclear DNA from bone fragments found at each grave site and compared them to DNA samples provided by some of Nicholas II and Alexandra's relatives. They also assessed DNA isolated from one of the Czar's bloodstained shirts, a museum piece saved from an attempt on his life in the late 1800s.

Whereas getting DNA from bone fragments found at the first grave site was relatively straightforward, Rogaev told GenomeWeb Daily News, the newly found bone fragments posed a "particular problem." That's because those bodies had been "burned and destroyed by fire and by sulfuric acid," he explained. Even so, they were eventually able to isolate and sequence DNA from even these badly damaged samples.

First, the team investigated the family's two mitochondrial lineages — that of Alexandra and her children and that of Nicholas II. Rogaev noted that they did not recover complete mitochondrial DNA sequences from the remains, but rather, assembled sequences from short fragments of mitochondrial sequence.

Since Empress Alexandra was Queen Victoria's granddaughter, the researchers compared mtDNA from her presumed remains with complete mitochondrial sequences from two of Queen Victoria's living relatives. They found that individuals from two branches of Queen Victoria's lineage — a great-great-granddaughter and a great-great-great-great-granddaughter — had identical 16,571 base pair mtDNA sequences.

The remains believed to be those of Alexandra and her children (three at the first grave site and two at the second grave site) all had the same sequence too, dubbed the "Queen Victoria" mitotype. On the other hand, when researchers searched a database containing 70,000 random individuals they didn't find anyone else with the complete Queen Victoria mtDNA sequence.

As expected, mtDNA from bone fragments believed to belong to Nicholas II had a different mitotype. While Nicholas II appears to have had a relatively common hypervariable region, the team found that his complete 16,569 base pair mitochondrial sequence only matched samples obtained from his maternal relatives but did not match complete mtDNA sequences in any available databases.

Meanwhile, Y-chromosome short tandem repeat sequence from DNA found in the putative Nicholas II and Alexei remains matched Y haplotypes detected in Nicholas II's second cousins. Again, the sequence was not found in American or Russian databases.

And, the authors noted, DNA analyses of bone samples from the male and female remains found at the second site "clearly indicate that the newly found bone specimens may belong to Prince Alexei and one of the daughters of the Imperial family."

During their analysis, the team stumbled upon a new resource: a shirt from the Hermitage Museum stained with Nicholas II's blood from an attempted assassination in Japan in 1891.

"I don't think that many people knew about [the shirt]. That was very interesting" Rogaev said. "I really did not count on that sample initially at all." But by testing several extracts from the bloodstained shirt, he explained, they isolated some DNA with mitochondrial, autosomal, and Y-chromosome DNA markers identical to those in Nicholas II's remains.

Overall, the team concluded, DNA evidence indicates that Nicholas, Alexandra, and all five of their children were killed in 1918 and buried in two separate graves a few dozen meters apart.

While Rogaev acknowledged that there have been persistent rumors that one or more Romanov children might have eluded the fate that befell the rest of the Russian royal family, he said DNA evidence shows otherwise. "That is what our study shows," he said. "That no one escaped."

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