Anthro Notes – Nov. 22/12
- Use of mtDNA in historical cases: For forensics, mtDNA has several advantages over nuclear DNA
when using degraded tissues and shed hairs – 1. High copy number per cell = greater probability of
getting DNA from degraded samples. 2. Maternal inheritance only (no recombination of gender lines
as is found with nuclear DNA). 3. Higher but generally stable mutational rate (10x greater) found in
the noncoding region, which is composed of hypervariable regions 1 and 2. Comparisons are made
easy by comparing the CRS differences between exemplar and evidentiary samples.
- The mitochondrial genome was fully sequenced in 1981 by Anderson. It has 16,569 bases. RCRC
(revised Cambridge reference sequence) was verified by Andrews in 1999. HV1 and HV2 are the
regions generally used in forensic cases. mtDNA has a high copy number. Nuclear DNA has 2 copies
per cell. It is inherited from both parents and is unique to an individual. mtDNA has more than 1000
copies per cell. It is maternally inherited and is not unique to an individual. Maternal inheritance of
mtDNA makes it easier to trace people over time among maternal relatives. Genealogical sleuthing is
difficult. HV1 (16024-16365) and HV2 (73-340) are the regions used to distinguish between
maternally related individuals. The high mutation rate is still stable over many generations (~30
- Comparing two samples using mtDNA hypervariable regions relative to the CRS: The two samples
have 5 mutational differences in the HV1 and HV2 regions relative to the CRS sequence. As well, all of
the mutations are the same. They are likely from the same mtDNA maternal lineage (in this case they
are siblings). In forensics, when a missing person is represented by bone and the suspected
individual has a maternal relative, the results of the evidentiary sample and the relative are
compared. If they match you cannot exclude this individual from being from that mtDNA lineage.
- Determining the probability of a match: If samples match (share the same mtDNA polymorphisms)
they are then compared to the FBI database. There are currently 4839 profiles in the database. If the
profile you are testing is in the database, use “n” from the racial category in which it is found. If the
profile is unique, use the total database and calculate the 95% confidence interval. You can also
produce a match using Bayesian stats and likelihood ratios.
- Problems with mtDNA analysis: mtDNA is very prone to contamination because of high copy number
and PCR selectivity (this requires a clean lab facility). It has low probative value compared to STRs
(usually 1/1000-2000 at the 95% confidence interval).
- Contamination controls: There are stringent procedural controls to prevent modern contamination.
Careful laboratory practices include experiments are carried out in a clean lab facility, protective
clothing is worn, and sterilization of equipment and reagents occurs. Negative controls are used. Labs
are dual-throughput and unidirectional.
- Romanov story: On July 16, 1917 at the height of the Bolshevik revolution in Russia, Czar Nicholas II,
Czarina Alexandra, four daughters (Olga, Maria, Tatyane, and Anastasia) and one son (Alexei) were
executed by firing by their Bolshevik captors. There were four additional victims including the family
physician Dr. Botkin. Six months later, Russian investigator Nicholas Sokolov recovered valuable
evidence from a gravesite, but found no skeletons (he hypothesize that evidence was destroyed). In
April 1989, Russian filmmaker Geli Ryabov announced that he knew where the Romanovs were
buried. He and a geologist found the grave 10 years earlier and retrieved three skulls. In 1991, Boris
Yelsin authorized the exhumation of a mass grave and 1000 bone fragments were assembled into five
females and four males. Two of the deceased were missing (the bodies of Alexei and a female were
allegedly burned). Russian scientists concluded that Tsarevich and Maria were missing. Dr. Maples, a
forensic anthropologist from Florida, believed that the grand-duchess Anastasia was missing. In
1992, research began on DNA work at the FSS conducted by Dr. P Gill to solve the mystery using nDNA and mtDNA. Five individuals were clearly related and three were female siblings. A sample of
mtDNA suspected to belong to Alexandra matched a sample provided by her grandnephew the Duke
of Edinburgh. In 1994, the body of grand duke Georgij Romanov (28-year-old brother of Czar
Nicholas who died of TB in 1899) was exhumed and sampled (part of the tibia and femur).
- Anthropological analysis: William Maples, a forensic anthropologist from Florida, used skeletal
evidence, dental records, and computer images of faces and claimed to have identified Czar Nicholas
II, his wife Alexandra, three of their children, three servants, and the family doctor. There was some
disagreement between Maples and the Russian scientists as to the identity of two of the other bodies.
The Russians thought that the missing female was Maria, but Maples insisted it was Anastasia.
- DNA analysis: In Sept. 1992, Pavel Ivanov carried samples to England to test them at Forensic Science
Service lab (FFS). The FSS was also involved in the first voir dire in R. vs. Morin. DNA testing of
several STR loci and amelogenin was done to test relationships among the nine individuals. mtDNA
analysis was conducted to confirm that the family was indeed the Romanovs. The remains of
Nicholas’ brother were sampled because of heteroplasmy. STR genotypes from the nine skeletons
were grouped together. A postulated pedigree was created. The children have alleles that are
inherited from their parents. The alleles from the excluded group included the three servants and the
doctor. Sex testing identified four males and five females. The son Alexei and one daughter
(Anastasia) were missing. This does conclusively identify them as the Romanovs. The Czar’s living
nephew Tikhon Kulikovsky refused to cooperate and would not provide a sample to identify the
Czar’s remains. Later, two other distant relatives Xenia C-Sfiri and the Duke of Fife agreed to help.
Their mtDNA matched each other and it also matched the sample believed to be from Czar Nicholas II
at seven mtDNA loci. At mtDNA locus 16169 there was heteroplasmy with the Czar’s sample (T/C).
The living relatives had base T at this locus. This heteroplasmic problem necessitated exhuming the
remains of Nicholas’ brother. When there is one base difference between an exemplar and an
evidentiary sample, the result is deemed a weak match. In the case of heteroplasmy, the result is a
stronger weak match. Heteroplasmy is where two distinct populations of mitochondria exist in the
same individual. The rate of heteroplasmy was found to be 70%. The work was done at the armed
forces DNA identification laboratory (AFDIL). Georgij Romanov’s mitotype matched Nicholas’ exactly,
including the heteroplasmic site. This makes the RMP even stronger. This was conclusive evidence
that the bodies found were the Romanovs.
- The Romanovs and the Anna Anderson case: At the same time as the research was being conducted, a
woman Anna Anderson claimed to be the missing Anastasia. Anna appeared in Russia in 1920 after
WWI and was hospitalized after attempting to commit suicide. She claimed to have amnesia. Those
caring for her noted how she resemble Anastasia (rumours about her survival were rife in post-war
Europe). Anna Anderson married an American, moved to Virginia, and died in 1984. A supporter of
the hypothesis that she was Anastasia was the grand-daughter of the Romanov’s doctor. She
(Schweitzer) obtained a 20-year-old paraffin-imbedded tissue sample of Anna from a Charlottesville
hospital, but it was not admissible. But there were also hair samples. Her mtDNA was compared with
the Romanov data and it was an exclusion. The paraffin tissue was later analyzed and it matched the
hair sample data.
- Conclusion: Despite some recent challenges to the results, most scientists feel that the results are
authentic. Research shows that nuclear DNA can be extracted from material less than 100 years old.
But nuclear DNA is not ideal for showing family lineages because of biparental inheritance. This case
shows the value of mtDNA for maternal lineage reconstruction in historic cases. Heteroplasmy can
actually increase RMP bec