DNA profiling has apparently laid to rest the last remaining mysterysurrounding the massacre of Russia's deposed Czar Nicholas II andhis immediate family in 1918. Or has it?

In July 1991, two amateur Russian historians excavated the bones ofnine individuals from a shallow grave near the city of Ekaterinburg,(once re-named Sverdlovsk) where the executions took place. Dentaland medical records strongly suggested that five of the nineskeletons belonged to Czar Nicholas Romanov, his wife, the CzarinaAlexandra, and three of their four daughters. Remains of the teen-agecrown prince Alexis and his 17-year-old sister, Princess Anastasia,were missing.

DNA sex-testing, short-tandem-repeat (STR) chromosomal analysisand mitochondrial DNA sequencing were performed in 1993 byforensic scientist Peter Gill and his associates at the British HomeOffice's Forensic Science Service, jointly with molecular biologistsat the Russian Academy of Sciences. Their conclusion "supports thehypothesis that the remains are that of the Romanov family." (SeeBioWorld Today, July 14, 1993, p. 1.)

Unlike chromosomal genes, mitochondrial DNA (mtDNA) isinherited only through one's female parents. Prince Philip, the Dukeof Edinburgh, Queen Elizabeth II's consort, is a maternallydescended grand-nephew of the Czarina Alexandra. He provided asample of his blood to the forensic scientists, which enabled them toconfirm that all three girls were siblings, and daughters ofAlexandra.

Two living, maternally descended relatives of Nicholas's maternalgrandmother had mtDNA sequences consistent with that of thepresumed Czar's skeletal remains.

A young woman claiming to be Anastasia surfaced in Germany circa1920, and for the next half century insisted, in vain, on her born rightto inherit the Romanov fortune, deposited in Swiss banks. Known asAnna Anderson, in 1968 she married John E. Manahan, a professorof history at the University of Virginia, in Charlottesville.

Anna Manahan underwent bowel surgery in 1979 at the MarthaJefferson Hospital in Charlottesville; she died in 1984. Nearly adecade later, a Charlottesville attorney, Richard Schweitzer, privatelycommissioned Peter Gill's Forensic Science Service in Britain to testsamples from her excised intestinal tissue for genomic homologywith known Romanov family descendants. Gill in turn invited theU.S. Armed Forces Institute of Pathology in Washington, to share inthe tissue-testing project.

This month's Nature Genetics describes the concordant DNAprofiling at both the U.K. and U.S. labs. Their paper, titled"Establishing the identity of Anna Anderson Manahan," alsomentioned "six hairs said to have come from Anna Anderson,analyzed by the Department of Anthropology, Pennsylvania StateUniversity," with its unpublished analytic procedure "in the press."

That hair analysis took place last fall. How it happened to happen isa story in itself, best told in the words of graduate student TerryMelton, who did the actual work in the Penn State mtDNA lab ofMark Stoneking. Both are co-authors of the current Nature Geneticsarticle.

"An amateur historian named Susan Burkhart, who lives in Durham,N.C.," Melton told BioWorld Today, "had a great interest in AnnaAnderson, and had access to the books from Jack Manahan's estate.[He died in 1990.] Burkhart had learned that professor Manahan hada habit of keeping locks of his wife's hair to give to her admirers.Apparently, she had a number of very ardent supporters over theyears, who believed that she was indeed the Grand DuchessAnastasia."

When Burkhart opened one book, Melton continued, out fell anenvelope with six hairs in it. "Fortunately," she observed, "the hairshad very nice roots on them, so we were able to do our analysis."Schweitzer told BioWorld Today that these were probably hairspulled out by a hair brush; that Manahan collected only cut locksfrom Anna's coiffure.

Burkhart told one of Anna's several biographers, Peter Kurth, abouther discovery, and because "Dr. Stoneking has quite a reputation indoing work on mtDNA analysis," Melton said, "he was contacted byKurth." Knowing of Melton's interest, Stoneking offered to let hertest the six hairs herself.

"We analyzed each one individually," she recounted, "by digestingthe hair with proteinase K, extracting the DNA and purifying it byethanol precipitation.

"Then we had genomic DNA. We used polymerase chain reaction toamplify just the two hypervariable regions of the mtDNA, which wethen sequenced. We needed to use primers that were reasonablyclose together, about 150 base pairs apart. We did the standarddideoxy sequencing reaction manually, because we do not have anautomatic sequencer."

Peter Gill and his associates at the Forensic Service lab inBirmingham, U.K., published their Anna Manahan intestinal-tissue-derived hypervariable mtDNA sequences in 1992 (Electrophoresis,13:173-175). These corresponded to the Ekaterinburg remains and tomtDNA from Britain's Romanov-related Prince Philip.

"When we reported our results to Peter Kurth," Melton recalled, wehad to add the caveat, `If indeed these are the hairs of AnnaManahan.' We had no way of knowing. The chain of custody onthose hairs was not very good, but the exciting thing for us was whenour results were announced, and Peter Gill contacted us. He asked uswhere our sequence was DD and it was a perfect match."

Although the tissue and hair samples matched each other, they didnot prove Anna's claim. As the Nature Genetics article concluded:"The samples could not be associated with a maternal relative of theCzarina or of His Royal Highness Prince Philip." n

-- David N. Leff Science Editor

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