A Royal Mystery Solved

DNA sequencing is usually associated with futuristic medical tests, but in one fascinating saga, it has explained a long-standing mysteryñthe fate of the Romanov family in Russia.

Determining the nucleotide base sequence of specific DNA molecules is a powerful tool with which to establish identities and relationships. The more closely two individuals are related, the more alike their DNA sequences will be.

DNA is sequenced by cutting it into pieces that differ in length by one base and labeling each terminal base with one of four fluorescent tags (A, C, T or G). An instrument manufactured by the Applied Biosystems Div. of Perkin-Elmer Corp., the 373A Automated DNA Sequencer, detects the fluorescence pattern of a DNA molecule, displaying it as a series of fluorescent peaks from which the inferred base sequence can be read. This technique was crucial in following the fate of the Romanovs, but in an unexpected way.

The mystery begins in July 1918, a year after the Bolshevik revolution ended the familyís rule. On one night, Czar Nicholas II, the Czarina Alexandra and their four daughters and one son, plus the family physician and three servants, were led to the cellar of the Ipatiev House in Yekaterinburg in Siberia. They were all shot, and those not dying quickly enough were bayoneted.

Some months later, a Russian investigator named Nikolai Sokolov intensely studied the royal disappearance, compiling seven volumes of evidence. He reported that the bodies had been trucked from the execution site and hacked up, the faces destroyed with sulfuric acid. Then they were burned and the ashes laid to rest in a shallow grave. And so the story went for decades.

In another July, in 1991, two amateur historians, Alexander Avdonin and Gely Ryabov, claimed to have uncovered a shallow grave 20 miles from where the Romanovs were believed to have been killed. President Boris Yeltsin ordered an exhumation of the remains.

The Russian governmentís official forensic investigation soon revealed that the nearly 1000 bits of bone at the site could be assembled into nine semi-skeletons. The sizes of the bones suggested that three were children, and gold, platinum and porcelain in some preserved teeth indicated royalty. Stab wounds and bullet holes supported the execution tale, and absence of facial bones supported the acid face-destroying story. So far, so good. Then the government sought DNA evidence, and the case fell to DNA experts Pavel Ivanov of the Russian Academy of Sciences and Peter Gill of the Forensic Science Service in Birmingham, UK.

First, the DNA sleuths determined which hones came from males and which from females by identifying the telltale Y chromosome indicating maleness. Then they delved into the DNA of mitochondria, the structures within cells that house the energy reactions. Mitochondria are passed from mothers to offspring and not from fathers because they are not present in the part of the sperm that enters the egg. Therefore, tracing mitochondrial genes reveals maternal lineages. Such analysis showed that the woman whom researchers suspected to be the Czarina because of her regal dental work was indeed the mother of the three children whose bones were found at the gravesite. A match between the womanís DNA and that of a known royal relative, Prince Philip, established her identity.

Meanwhile, DNA work elsewhere disproved the claim of a woman named Anna Anderson that she was Anastasia, one of the Romanov children whose bones were not found at the gravesite. Anderson had moved from a mental hospital in Berlin in 1921 and settled in Charlottesville, Va., insisting until her death in 1984 that she was the missing Romanov daughter. DNA analysis of blood taken for a test in Germany many years earlier, on tissue taken for a cancer test in Charlottesville and on hair that a historian found in one of Annaís books all proved not to be of Romanov extraction.

Now that the Czarina had been linked to the royal family, it seemed likely that a male skeleton with nifty dental work was the Czar. Certainly, government investigators thought, DNA analysis would confirm what everyone expected. They were in for a surprise.

The problem centered on nucleotide position 16169 of a mitochondrial gene that is highly variable in sequence among individuals. The fluorescence peak for the bone cells of the purported Czar, which should have been one color, was twoñabout 70 percent blue, indicating cytosine (C), and 30 percent orange, indicating thymine (T). Skeptics at first suspected a laboratory error or contamination, but when several independent analyses consistently yielded the same perplexing result, genetic researchers realized that this historical case had led them to a phenomenon not seen before in human DNA samples. The bone cells obviously harbored two populations of mitochondria, one type with C at this position, the other with T.

A DNA quirk would be acceptable as proof of identity as long as it showed up in royal relatives. But it didnít, at least at first. The supposed Czarís odd bone cells confused the so-far elegant historical reconstruction because cells of a living blood relative of the Czar, Countess Xenia Cheremeteff-Sfiri, revealed only T at nucleotide site 16169ña lone but high orange "T" peak to the Czarís bicolored twin peaks. However, DNA between Xenia and Nicholas matched at every other site. Xenia is the great-granddaughter of Czar Nicholas IIís sister.

Another living relative, the Duke of Fife, the great-grandson of Nicholasí maternal aunt, matched Xenia at the famed 16169 site. A closer relative, Nicholasí nephew Tikhon Kulikovsky, declined to participate in the DNA quest, citing anger at the British for not assisting the Czarís family during the Bolshevik revolution.

So ended this second chapter of the Romanov tale, with the Russian government unsatisfied with how the mystery had been solved. It would take an event in yet another July to clarify matters.

The Russian Federation government asked a research team to look to Nicholasí brother, Grand Duke of Russia Georgij Romanov, to see if another blood relative had the peculiarity at site 16169ñthis would prove that the remains were indeed those of the Czar. Georgij had died at age 28 in 1899 of tuberculosis. He was exhumed from St. Peter and Paul Cathedral in St. Petersburg in July 1994, and the researchers set to work analyzing the DNA in his leg bones. The team consisted of Pavel Ivanov, who had done the earlier work, and a group led by Thomas J. Parsons of the US Armed Forces DNA Identification Laboratory in Rockville, Md.

Happily for all concerned, except perhaps for the doomed royals, Georgijís mitochondrial DNA had the same double-base site as the remains, which were therefore those of Czar Nicholas II. The researchers calculated the probability that the remains are truly those of the Czar, rather than resembling Georgij by chance, as 130 million to one. They conclude in their report in the April issue of Nature Genetics, "Given that anthropological and circumstantial evidence was also considered conclusive for the identification of the Romanovs, there now appears to be no reasonable scientific objection to accepting the authenticity of the remains."

Thanks to DNA analysis, the murdered Russian royal family can finally rest in peace.

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