No heteroplasmy at base position 16169 of Tsar Nicholai Ills mitochondrial DNA
T.* Nagaiu, T. Okazaki*-*, M. Shlmadzu'and V. L. Рорov
Peter Gill et al. (Nature Genetics, 6:130-135,1994) and Pavel L. Ivanov etal. (Nature Genetics, 12:417-420,1996) analyzed the DNA sequence of the hypervariable region (HVR) of mitochondrial DNA (mtDNA) extracted from Tsar Nicholai ll's bone, and reported that there was a heteroplasmy (C/T) of cytosine (C) and thymine (T) at base position 16169.
In order to confirm their finding, we analyzed, using the usual method, the mtDNA sequences of Tsar Nicholai ll's sweat stain, hair/nail/bone of Grand Duke Georgiy Alexandrovich (Tsar's brother), and a blood sample obtained from Tihon Nikolaevich Kulikovsky Romanoff (son of Tsar Nicholas ll's youngest sister, Grand Duches Olga Alexandrovna) before his death and stored at -80. The following four primers were used:
HVRI: F15989 (s'-CCCAAAGCTAAGATTCTAAT)
HVR: F29 (5'-CTCACGGGAGCTCTCCATGC)
We obtained results similar to those reported by Peter Gill et al. and Pavel L. Ivanov et al., except that we did not find heteroplasmy (C/T) at base position 16169, the finding to which they attached the greatest importance in their identification test. The base at position 16169 was С in all samples used in our test. In addition, although we searched for T by restriction enzyme digestion using a primer into which a restriction enzyme recognizing site was introduced, no was found in any of the samples tested.
These results lead us to the conclusion that Tsar Nicholai IP mtDNA has no heteroplasmy (C/T) at position 16169, but does have homoplasmic cytosine. Thus arises the question of whose bone was examined by Pater Gill et al. and Pavel L. Ivanov et al. Who is buried in the grave of the Peter and Paul Cathedral at St. Petersburg?
Finally, the assistance of Mrs. Olga Nikolaevna Kulikovsky Romanoff, who kindly provided us with Tihon Nikolaevich Kulikovsky Romanoff's blood sample, is gratefully acknowledged.