Six differences in HV1 is a substantial divergence. In forensic genetics, even a single positional difference in the hypervariable region is typically considered an exclusion when comparing two individuals who should share the same maternal line. The logic is that if Person A and Person B are supposed to be mother and daughter — or share any other close maternal relationship — their mtDNA profiles should be identical (or differ by at most one position, accounting for the rare possibility of a de novo mutation). A six-position difference is far beyond the margin for error or mutation.
When Prince Philip donated blood as a reference sample, his mtDNA represented the Romanov maternal line: he and Tsarina Alexandra shared the same maternal ancestor (Princess Alice of the United Kingdom, who was Queen Victoria’s daughter). Philip’s mtDNA was thus an accurate reference for what Anastasia’s mtDNA should have looked like.
The six-position difference between Anderson’s profile and Philip’s profile is the biological statement of the exclusion: whoever Anderson’s maternal ancestors were, they were not the maternal ancestors of Tsarina Alexandra and her children.
A separate confirmation came from the heteroplasmy found in Nicholas II’s remains. Heteroplasmy is the condition of having two slightly different versions of the mitochondrial genome in the same individual. It is rare, and its presence in a tested sample is distinctive enough to serve as an additional marker. Nicholas II showed a specific heteroplasmy at position 16169 in his mtDNA. This unusual variant was confirmed by testing the exhumed remains of his brother, Grand Duke George, who died in 1899 — supporting the identification of the 1991 grave remains as Nicholas II. The heteroplasmy pattern was not present in Anderson’s profile, which was another dimension of difference between her and the Romanov family.