Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters.

[1]  Walther Parson,et al.  EMPOP--a forensic mtDNA database. , 2007, Forensic science international. Genetics.

[2]  Suni M. Edson,et al.  Naming the Dead - Confronting the Realities of Rapid Identification of Degraded Skeletal Remains. , 2004, Forensic science review.

[3]  L. Zhivotovsky Recognition of the remains of Tsar Nicholas II and his family: a case of premature identification? , 1999, Annals of human biology.

[4]  T. Parsons,et al.  Application of Low Copy Number STR Typing to the Identification of Aged, Degraded Skeletal Remains * , 2007, Journal of forensic sciences.

[5]  T. Melton,et al.  Mitochondrial DNA Heteroplasmy. , 2004, Forensic science review.

[6]  R. Massie The Romanovs - The Final Chapter , 1995 .

[7]  P. Lincoln DNA recommendations--further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. , 1997, Forensic science international.

[8]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[9]  Ongoing Controversy over Romanov Remains , 2004, Science.

[10]  W. Parson,et al.  ‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples , 2008, International Journal of Legal Medicine.

[11]  Yin Shen,et al.  The development of reduced size STR amplicons as tools for analysis of degraded DNA. , 2003, Journal of forensic sciences.

[12]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[13]  Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples. , 2009, Forensic science international. Genetics.

[14]  P. Gill,et al.  Identification of the remains of the Romanov family by DNA analysis , 1994, Nature Genetics.

[15]  J Buckleton,et al.  An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. , 2000, Forensic science international.

[16]  Peter Gill,et al.  Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci , 2005, International Journal of Legal Medicine.

[17]  M. Hofreiter,et al.  Assessing ancient DNA studies. , 2005, Trends in ecology & evolution.

[18]  P. Ivanov,et al.  Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II , 1996, Nature Genetics.

[19]  Jerilyn A. Walker,et al.  Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification. , 2005, Analytical Biochemistry.

[20]  Edward M. Robinson,et al.  Integrated DNA and Fingerprint Analyses in the Identification of 60‐Year‐Old Mummified Human Remains Discovered in an Alaskan Glacier , 2010, Journal of forensic sciences.

[21]  A. Carracedo,et al.  DNA recommendations , 1997, International Journal of Legal Medicine.

[22]  W. Parson,et al.  A new “miniSTR-multiplex” displaying reduced amplicon lengths for the analysis of degraded DNA , 2006, International Journal of Legal Medicine.

[23]  T. Parsons,et al.  High efficiency DNA extraction from bone by total demineralization. , 2007, Forensic science international. Genetics.

[24]  Walther Parson,et al.  A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. , 2007, Forensic science international. Genetics.

[25]  P. S. White,et al.  Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remains , 2004, Annals of human biology.