Presentation by:
Megan Latulippe
Jennifer Nagel
Moyin Adesanya
Isabel Orach
Raahyma Ahmad

The Romanov Family History, Politics, and
Miscellaneous
“The Royal Disease”: Hemophilia
Main Paper
Secondary Article of Interest
Author and People of Importance
Modern Applications of Techniques
References

The Romanov tzarist dynasty was the last reigning dynasty of Russia.
In 1894 Nicholas II ascended to the throne marking the beginning of the fall of the Romanovs. Amongst the crushing responsibilities as rules and growing political unrest, Nicholas and Alexandra were distraught parents faced with maddening helplessness against their son, and only heir’s, debilitating haemophilia.

Tzar Nicholas II (1868-1918) ( 1894-1918) age 50
Tzarina Alexandra Fyodorovna (1872-1918) age 46
Grand Duchess Olga Nikolaievna Romanov (1895-1918) age 22
Grand Duchess Tatiana Nikolaievna Romanov (1897-1918) age 21
Grand Duchess Maria Nikolaievna Romanov (1899-1918) age 19
Grand Duchess Anastasia Nikolaievna Romanov (1901-1918) age 17
Tzarevich Alexei Nikolaievich Romanov (1904-1918) age 13

It is difficult to objectively view the case of the Romanovs.
On one hand Tzar Nicholas II made many errors in the eyes of his people, failing the common public and leaving them poor, hungry, and reeling from the devastating effects of war.
While on the other hand, quick to react revolutionist over took the distraught rulers by force, culminating in the violent and bloody murder of the Tzar, Tzarina, their five children, and four servants.
Demonstrations during
July Days ending in
Provisional Government soldiers opening fire upon civilians
(Petrograd / Saint
Petersburg – 1917)

The Russian Revolution of 1917 can be viewed as two separate revolutions. A number of driving factors contributed to the Russian
Revolution including widespread poverty and famine, unrest between working and social classes, as well as the battle for power over Russia between the Tzar and his opposing political parties. The affairs of
Russia were drastically worsened by the losses of war.
Overall the February Revolution, having occurred during
February and March of 1917, concluded with the forced abdication of
Tzar Nicholas II. Although a provisional government was established calm amongst the people was not established. In October and
November the civil and political unrest broke out again, known as the
October Revolution, leading to the Bolshevik Party seizing power of the government. Unknown to the rest of the world this was the beginning of Soviet Russia.

Nicholas II
Coronation
Bloody
Sunday
May 14, 1896 Jan 1, 1905
Formation of
Duma
Aug 19, 1905
February
Revolution
Provisional
Government
Formed
Feb/Mar
1917
(Mar 16, 1917)
October
Revolution
Bolshevik Party seize control of governme nt
Oct/Nov 1917
Romanov
Execution
Jul 17, 1918
Feb 4, 1904 Feb 1905 Aug 1, 1914 Mar 16, 1917
Japan Declares
War
February
Riots
Germany
Declares War
(WWI)
Nicholas II
Abdication
Jul 31, 1917
(Apr/May 1918)
Romanov family exiled to Tobolsk
Romanovs exiled to Ural region (Siberia)

Under the control of Lenin, the Bolshevik Party exiled the
Romanovs to Yekaterinburg city in a dreary area of the
Ural Mountains of Siberia. Under the guise of protection from revolutionists, the Romanovs were imprisoned in the
Ipatiev House. A house that would become known as the
“House of Special Purpose”. For months the royal family, their attending physician, and three servants lived under constant threat.

Graphic Design of Ipatiev House http://www.romanov-memorial.com/Outside.htm

Approximately 2am March 17 1918 the Romanov family was woken from their beds, along with their servants, and rushed to a cellar room of the
Ipatiev house. Once again under the false pretenses of safety from growing riots the eleven prisoners were herded like cattle. Lenin had decided the risk of the Romanov’s rescue was advancing. Yakov Yurovsky, head of the
Romanov’s guards and firing squad, proceeded to carry out his orders.
While the confused and helpless Romanovs huddled together for their last moments Yurovsky and his men opened fire. Written accounts from
Yurovsky himself indicated that the firing went on for several minutes with confusion erupting through out his men as the cellar room filled with smoke and the deafening sounds of gunshots and screams.

Four Brothers Mine
To hide their crimes and ultimately to prevent
Romanov sympathizers, Yurovsky and his men transported the brutalized bodies to the
Four Brother mine near the Koptyaki Forest in hopes of concealing them. After the bodies were stripped, searched for jewels, and further violated they were tossed down the shallow pit with grenades.
Yurovsky, encountered with unforeseen circumstances and paranoia, forced his men to return to the Koptyaki Forest to retrieve the bodies and further dispose of them. Attempts to dismember and burn the two smallest bodies failed and as a result of anxiety and panic the remaining bodies were further disfigured with acid and buried in a shallow grave separate from the burnt bodies.
Grave 1 with 9 sets of remains

Long after the tragic murders, the fate of the Romanovs was concealed. Statements circulated assuring the family’s survival were followed by statements explaining the execution of only the Tzar. Once the knowledge of their deaths was no longer secret Lenin claimed radical
Jewish conspirators were responsible. To further evade blame it was also declared that Lenin was unaware of the plan hatched by his men and played no part in the orders himself. As Russia descended further into
Soviet control the truth of the Romanovs began to disappear. Many official documents concerning the case were destroyed or locked away. The affairs of Soviet Russia became so corrupt and secretive that the discovery of the first mass grave in 1979, suspected of containing the Romanov remains, was kept hidden until 1991. Researchers waited for the fall of the Soviet
Union in hopes that the remains would be examined by outside, unbiased sources.

Even after genetic analysis of the remains found within Grave 1 much speculation remained as to whether the missing royals had finally been found. For starters two members of the Romanov children were missing; Tzarevich Alexei and a younger daughter – either Anastasia or
Maria. With the discovery of the second grave in 2007 and further genetic analysis it is believed that mystery has finally been solved.

Due to false statements given to the public assuring them of the
Romanov’s safety as well as rumours told by Yurovsky’s men concerning the difficulty of the actual execution a legend was born.
Over time many imposters have surfaced claiming to be surviving members of the Romanov family, none are more famous than the self-proclaimed Anastasia; Anna
Anderson. Anna – later identified as Franziska
Schanzkowaska of Poland – began her claims in the 1920’s and died still believing she was Anastasia.
Eugene Nicolaievich Ivanoff is one of the many
Tzarevich Alexei imposters. In 1927 Ivanoff began his claims. Combined with his knowledge of Russian court, his fluency in English, German, and French, and his apparent haemophilia he sparked much controversy.
Subsequent DNA tests performed after remains of the final two bodies were discovered proved such claims to be false and all to be imposters.

X-linked recessive disorder.
Must possess two recessive alleles in females, and one recessive allele in males to be afflicted with hemophilia.
Characterized by the inability to form blood clots.
Mutations in genes that code for coagulation factors XIII and IX.
Minor injuries results in internal hemorrhaging which could be fatal.

It all began with Queen Victoria of England, whom was a carrier for hemophilia B.
Became what was known as “The Royal’s Disease”

Tsarina Alexandra was a carrier for hemophilia B.
Unfortunately, this was passed on to Alexei, who suffered from the terrible hereditary disorder!

Early 19 th century when the Prince of Coburg married a Hungarian
Princess named Antoinette de Kohary.
A monk of the Kohary family, envied the wealth inherited by the happy couple and cursed future generations of Coburgs with the terrible disease.
Obviously, today we know that hemophilia is a genetic disorder that affected the royal family, and not a curse.
Though it makes for a good story!

Initially analyzed extracts of degraded DNA from skeletal bone specimens of Tsarina Alexandra.
Applied a multiplex target amplification and massively parallel sequencing (MPS) methods.
This allowed the retrieval and characterization of nuclear gene sequences.
Initial mutation screening of genes for blood factor VIII (F8), which contains 26 exons, and factor IX (F9), which contains 8 exons. These genes are both located on the X chromosome.
Multiple pairs of primers were constructed for short amplicons, because the DNA was limited in terms of quantity and quality. It was then combined with polymerase-chain reaction (PCR) in order to amplify the F8 and F9 genes.
Analyzed the genes using MPS in tandem with conventional sequencing. Note: MPS of the mtDNA was included as a control to ensure there was no contamination of unrelated DNA.

Results:
Found no evidence on non-synonymous, missense, or small insertion-deletion mutations in either F8 or F9 genes.
Detection of A to G intronic mutation located in 3 base pairs upstream of exon 4 in the F9 gene, that could be pathogenic.
Bioinformatics analysis predicts that the mutation shifts the open-reading frame of the F9 mRNA, leading to a premature stop-codon. The protein is therefore not completely translated and its function is disrupted.

Both wild-type and mutant alleles were detected in Alexandra (X H X h ).
Only one mutant allele discovered in Alexei (X h Y).
Determined that Anastasia was a heterozygous carrier (X H X h ).
Based on this information, how severe was Alexei’s form of hemophilia?
An in vitro exon trap assay was used alongside MPS, and it was discovered that 99.98% of the transcripts were generated by splicing at the mutant F9 allelic site. Therefore, Alexei possessed a severe form of hemophilia due to the fact that only 1% of F8 or F9 was functional.

X H
Y
X H
X H X H
X H Y
X h
X H X h
X h Y
First scenario: Mother is a carrier and is not afflicted by the disease.
Mother will pass on the X h to ½ of the children, ¼ of which are the daughters and ¼ of which are the sons.
All sons that received the X h will have hemophilia.
The daughters in this case are carriers, because they possess only one of the recessive alleles. They will pass this on to their children.
The daughters do not have hemophilia.

X h
Y
X H
X H X h
X H Y
Scenario 2: The Father has hemophilia.
X H
X H X h
X H Y
The father will pass on X h to all daughters, whom will be carriers.
Sons are not afflicted by hemophilia, as they receive the X H chromosome from the mother, whom is not a carrier.

X h
Y
X H
X H X h
X H Y
X h
X h X h
X h Y
Scenario 3: Father has hemophilia, and the mother is a carrier.
3/4 of the children will receive an X h .
½ of daughters will be carriers, while the other ½ will have hemophilia.
½ of the sons will be unaffected, while the other ½ will have hemophilia.

X h
Y
X h
X h X h
X h Y
Scenario 4: Both parents have hemophilia.
All children will have hemophilia.
X h
X h X h
X h Y

Hemophilia A:
The most common form of hemophilia.
Affects less than 1 in 10,000 individuals, or approximately
2,500 Canadians.
Characterized by a deficiency of factor VIII, which causes a clotting problem in the blood

Hemophilia B:
Affects around 1 in 50,000 people, or approximately 600
Canadians.
Known as Christmas Disease, due to Steven Christmas, the first person to be diagnosed with this form of the disease.
Characterized by a deficiency of factor IX, which slows down the clotting process.

At the time the only diagnosis was the observation of prolonged bleeding after bumping into objects or falling down.
Accidents that normally wouldn’t cause extreme internal hemorrhaging.

Alexei was carried around a lot to prevent bumping into things or falling down. The royal doctor would also bind Alexei’s wounds tightly.
Grigori Rasputin, a mystic, claimed to possess healing magic and as
Tsarina Alexandra was desperate to try anything she allowed the mystic to work on Alexei. Evidently, this did not work.
Due to the fact that very little was known about the disease, there were no solid treatments.
No medications were available at the time .

Blood tests are used to reveal what the levels of blood factors in the blood.
Three classifications for Hemophilia A and B:
Classification Level of Factor VIII or IX in Blood
Severe Less than 1% of normal
Moderate
Mild
1 to 5% of normal
5 to 30% of normal

Factor replacement therapy : “infusion (injection into the bloodstream) of factor VIII and IX concentrates to prevent or control bleeding” (Canadian Hemophilia Society, 2016).
Physiotherapy: used to ensure that joint movement is maintained and that muscles remain strong .
Desmopressin: a synthetic drug that is a copy of a natural hormone.
Useful in treating patients with mild or moderate hemophilia A. It acts by increasing the release of von willebrand factors which is thought to increase the release of factor VIII.

Cyklokapron and Amicar: Useful in treating both hemophilia A and B. It is a drug that helps hold a clot in place once it has formed, as it prevents the activity of the enzyme plasmin, which dissolves clots. These do not help form the clot, therefore it is not a substitute for desmopressin or factor infusion therapy.

Development of inhibitors: the body’s immune system sees the factor as a foreign invader and destroys it.
Damage to joints as a result of repeated bleedings
Blood-borne infections such as HIV, Hepatitis A and C were transmitted by factor concentrates in the past

With proper treatment a person living with hemophilia can still live a relatively normal life in today’s society.
Participate in sports, such as: swimming, golf, volleyball, and skating to name a few.
This helps keep muscles strong and joints mobile!

Peter Gill et al., 1994

Dental Evidence:
Some of the bodies had gold, platinum and porcelain dental work, an indication that at least some of the remains were aristocrats.

• All the skeletons showed evidence of violence and mistreatment before death.
• Some of the skulls had bullet wounds.
• All the bodies were found in the correct location.
• All the bodies were estimated to be the correct age and sex.
• Bayonet marks were found.
• Facial areas of the skulls were destroyed.

1.
Sex Testing
-Used to determine the sex of bones found.
2.
Short Tandem Repeats Analysis
3.
-Used to establish whether a family group was present.
Mitochondrial DNA (mtDNA) Analysis
-Used to determine relatedness with known maternally related descendants of the Romanov Family.

The sex of the bones was determined by amplification of a portion of the X-Y homologous gene: Amelogenin.
• Amelogenin is the name of a series of proteins involved in the development of enamel.
• Differences between the X chromosome (AMELX) form and Y chromosome
(AMELY) form of the Amelogenin gene enables it to be used in the sex determination of unknown human samples.
• The X chromosome (AMELX) form of the amelogenin gene contains a 6bp deletion at intron 1 relative to the Y chromosome (AMELY) form.
• This can be detected using primers which bind to the first intron region of the amelogenin gene on the X and Y chromosome, followed by Polymerase
Chain Reaction (PCR) to amplify the intron region and gel electrophoresis to separate the regions.

• Methods
Using a single pair of primers spanning part of the first intron,
106 bp and 112 bp PCR products were generated from the X and
Y homologues respectively, these were then resolved by agarose gel electrophoresis.
• Results
The results of DNA extracts from all nine skeletons confirmed the conclusions regarding their sex drawn from physical examinations of the bones
In total, four male and five female bodies were identifies using this method

The human genome is full of repeated DNA sequences which come in various sizes. Short Tandem Repeats are DNA regions with short repeats that are usually
2 to 6 base pairs in length.
An individual inherits one copy of an STR from each parent, which may or may not have similar repeat sizes. The smaller size of STR alleles make STR markers better candidates for use in forensic applications, in which degraded DNA is common. Short Tandem Repeat (STR) analysis can be used to demonstrate closer relationships of family groups consisting of siblings and their parents.

Methods
Five tetrameric loci : HUMTH01 10 , HUMVWA31 11 , HUMF13A1 12 ,
HUMFES/FPS 13 and HUMACTPB2 14 were amplified using the Polymerase
Chain Reaction from each of the nine skeletons.
In order to avoid mistyping, all samples were analyzed a minimum of four times and any homozygotes were analyzed a minimum of six times.
Results
All skeletal samples yielded amplification products corresponding to expected allele sizes.
The STR evidence demonstrated the presence of a family among the remains found in the mass grave

Figure 1: STR genotypes of the nine skeletal samples
• The observed genotypes of skeletons 3-7 exhibited patterns which would be expected in a family group.
• Skeletons 4 and 7 were the parents of children 3, 5 and 6.
• All other adults were excluded as possible parents.

Figure 1: Analysis of skeletal remains at STR locus HUMTH01
Blue bands are amplified HUMTH01 alleles
Red bands are internal electrophoresis size markers.

STR Analysis results support the hypothesis that bodies 3-7 were related
If the remains are those of the Romanovs, then the STR analysis and sex testing results indicate that one of the princesses and Tsarevitch Alexei were missing from the grave. This would support some historical accounts which indicate that two bodies were either burned or buried separately.
Alternatively, two individuals may have survived the massacre.
However, STR results cannot provide information that specifically identifies whether this is the Romanov Family. This can only be addressed by comparing mtDNA sequences with known maternal relatives.

The mitochondria is the powerhouse of eukaryotic cells. The human mitochondrial genome consists of 16,569 base pairs compared to the human nuclear genome which consists of 3.3 billion base pairs of DNA. The mitochondrial genome is circular and contains 37 genes that encode 13 proteins, 22 tRNAs, and 2 rRNAs. One mitochondrion contains dozens of copies of its mitochondrial genome and each cell can contain numerous mitochondria. Therefore, a given cell can contain several thousand copies of its mitochondrial genome. Mitochondrial mode of inheritance is strictly maternal, therefore mitochondria-associated disease mutations are also always inherited maternally.

• mtDNA contains two hypervariable regions located within the D-loop of mitochondrial
DNA. These regions are used in DNA testing because they are highly polymorphic therefore they can occur in several different forms.
• mtDNA is an even more powerful tool in forensic investigations because it is usual present at a high copy number in cells.
• It is also capable of surviving for prolonged periods compared to that of chromosomal
DNA.
• mtDNA analysis can be used effectively where victims and living descendants are separated by many generations.

Methods
Sequenced both hypervariable regions of mtDNA in all samples with the exception of skeleton 9 (a probable servant) for which sequence from nucleotides
16216 to 16360 was determined by manual sequencing from one strand only.
HRH Prince Phillip, the duke of Edinburgh (the grand nephew of unbroken maternal descent from Tsarina) provided blood samples for comparison purposes. All of the mtDNA sequences were the same.

There were no sequence differences observed between duplicate samples from the same individuals.
In general, 380 nucleotides were observed in the 1 st hypervariable region and 360 nucleotides were observed in the 2 nd hypervariable region from the amplified bone
DNA extracts.
Pairwise comparisons showed that six different sequences were present in the group which varied on average by 6 nucleotide and identical sequences were generated from the Tsarina Alexandra and the 3 children.

A comparison of mtDNA sequences of the proposed Tsar and two maternal relatives of unbroken descent from the
Tsar’s maternal grandmother showed that they had the same sequence as the Tsar with the exception of a single nucleotide at position 16169!
Further analysis of heteroplasmy at this position showed that it comprises of nucleotides C and T present at a ration of approximately 3.4:1

Heteroplasmy was observed in duplicate extracts from skeleton 4.
This was done by sequencing individual recombinant clones which were constructed by ligating PCR products into the plasmid pUC18 followed by transformation into E. coli cells.
Upon sequencing, 28 clones had C at nucleotides 16169 while 7 clones had T at
16169 giving a ratio of
4:1.

mtDNA sequences of the mother and grandmother (Louise of Hesse-
Cassel) of Tsar Nicholas II are not available so it is not known whether either woman exhibited this sequence heteroplasmy.
• In conclusion, STR Analysis and mtDNA sequencing have established that the remains are almost certainly those of the Romanov Family.
• The use of DNA Analysis techniques has provided a dramatic insight into the power of these techniques for use in solving historical questions.

2.
3.
1.
4.
While STR analysis was important for confirming that bodies 3-7 were related, it can’t tell us if these were the bodies of the Romanov family.
This is why mtDNA was so important.
The probabilistic analysis of mtDNA proceeded by first offering a few scenarios to consider:
Group R was that of the Romanov family, and group R’ was unrelated to the Romanov family.
These remains are completely unrelated to the Romanov family.
The group is another family related to the Romanovs (disproven because historical evidence showed there were no related families of the correct age or sex).
It was all a hoax! (Yeah, right).

5. Contamination?
Unlikely because:
(i) Identical results were procured from two different bones.
(ii) The negative control (no bone powder added) was tested in parallel with different bones, and therefore, for the presence of a contaminant to be considered, it must be observed in all samples, and not just the one.
(iii) Unlabeled bone samples were extracted, amplified, and sequenced in different laboratories.
(iv) STR results were not mixtures
(v) On average there is a 8.5 bp difference between white Caucasians in a population in the UK, therefore, the probability that just one base pair be different in two random sequences is p=0.014.

One of the major reasons the original paper was subject to heavy criticism is due to the fact that the supposed Tsar Nicholas II had mtDNA that was heteroplasmic. Therefore, the public claimed it simply could not be the Tsar who was found in the grave.
However, people in a population are known to have SNPs (single nucleotide polymorphisms).
Therefore what is the probability of the Tsar possessing a single mutation?

Calculated to be approximately 1/300 per generation. This however does not account for heteroplasmy, which may go undetected.
The mutation played an important role in the interpretation because the strength of the evidence depended upon whether or not the mutation was accepted.
This meant that the researchers were unsure of whether to include the mutation (weakening their findings) in the analysis or to forgo it (strengthening their findings).
They chose to perform two tests: one with the mutation and one without.

The likelihood ratio was used to analyse the DNA evidence which evaluates two competing scenarios:
LR= P(E|R)
P(E|R’)
In the numerator, the probability of the DNA evidence (E) is tested, given the alternative hypothesis (R) that the remains belong to the Romanovs.
In the denominator, the probability of the DNA evidence
(E) is tested, given the null hypothesis (R’) that the remains do not belong to the Romanovs.

Lower Bound Likelihood ratio: This ratio was taken while accounting for the mutation using the following equation:
LR= P(E|R)
P(E|R’)
This calculates the ratio of the probability of the DNA evidence given that the remains did belong to the Romanov’s.
For this calculation, the numerator includes the probability of a mutation occurring in the Tsar but not in any of the generations between him and living family members. The denominator therefore uses the probability of random individuals differing by only on base pair.
With this calculation, the evidence suggests that the remains are 70 times more likely to be Romanov’s than random individuals.

Upper Bound Likelihood ratio: This ratio was calculated, not taking into account the mutation using the following equation:
LR= P(E|R)
P(E|R’)
This again calculates the ratio of the probability of the DNA evidence given that the remains did belong to the Romanov’s.
However, for this calculation, the numerator evaluates the probability of the evidence, with the assumption that no mutations occurred. The denominator also changes, using random individuals to calculate the probability.
With this calculation, the evidence suggests that the remains are 80,000 times more likely to be Romanovs than random individuals.

Even with all of this evidence, people were STILL skeptical!
It wasn’t until a new article published in 2009 that confirmed the original findings to be true, that people started to accept the fact that the bodies were that of the Romanovs.

An article published in 2009 duplicated the techniques applied in Gill et al’s., original paper in order to confirm whether or not it was in fact the Romanov family’s DNA that was found in the grave.
This was done because the original article received heavy criticism questioning its validity.
Also, who doesn’t love a good mystery?!

Russians believed it was Maria missing from the mass grave, while
Americans believed it was Anastasia.
In the summer of 2007, a group of amateur geologists discovered bone fragments approximately 70m from the first grave.
In total, 44 bone fragments and teeth were recovered by expert archeologists of the Sverdlovsk Region’s Archeological Institute.
Based on the bone fragments collected, no less than two people were said to be among the discovered remains.
One of the remains belonged to a female. This was determined by visible sciatic notch dimensions. They estimated the age of the remains to be somewhere between 15-19 years old.
The sex of the second person was highly suspected to be male, once again based on the emerging broadness of the sciatic notch.
The biological age was estimated to be between 12-15 years old.

In order to prove the identity of the two sets of remains found within the 2007 grave mtDNA analysis was performed
Sample Origin Position within HVRI Region Position within HVRII Region
Alexandra
Male
(Alexei ?)
Female
(Anastasia ?)
16111
T
T
T
16357
C
C
C
263
G
G
G
315.1
C
C
C
Only HVR with variation from reference sequence noted mtDNA control regions within HVRI & HVRII were sequenced from each of the remains of Grave 2 as well as re-sequenced from the original remains from Grave 1 (Tzarina and four daughters). Results support Peter Gill and his results and examination revealed the two newly discovered remains were in fact children of the Tzarina.

When referencing the German and West Eurasian Databases no exact matches to the Tzarina’s mtDNA sequence were found. This greatly supports the assumption that the two new discovered remains are indeed the Tzarina’s children; Alexei and one of his sisters.

Bone fragments were studied by anthropologists who determined that one male and one female was present.
Anthropologists determined that fragment 146.1 belonged to a male whereas fragment 147 belonged to a female – both came from a femoral bone.
Autosomal STR testing for the amelogenin marker confirmed that fragment
146.1 was male and 147 was female.

Autosomal STR analysis for the second grave revealed allele sharing between the two separate individuals – DNA evidence indicates that the two are 5.6 million times more likely to be siblings than unrelated individuals.
Autosomal STR genotypes were developed for the first grave as well to confirm previous findings.
The exact same results as those presented in the first paper were produced.
Half allele sharing at all the autosomal STR genotype markers indicate that the remains found in the new grave are the children of Tsar and
Tsarina.

Performed to compare remains of Tsar and his son to a paternally related living descendant.
17 marker haplotypes of Tsar, Alexei and Andrew Andreevich Romanov show an exact match between all 3 individuals over all 17 markers.

Samples were tested independently at two labs and independently sent to Peter Gill to confirm that the data was in concordance.
The likelihood ratio was used to analyse the STR evidence which evaluates two competing scenarios:
LR=
In the numerator, the probability of the DNA evidence (E) is tested, given the alternative hypothesis (H
1
) that a relation exists.
In the denominator, the probability of the DNA evidence (E) is tested, given the null hypothesis (H
2
) that no relation exists i.e two random individuals are being tested.
The DNAView program was used for Likelihood ratio calculations.

The Sibship Index was calculated from performing the likelihood ration given:
Ho/Null hypothesis: The DNA samples from bones 147 and 146,1 indicate that the two individuals are unrelated individual.
Ha/Alternative hypothesis: The DNA samples from bones 147 and 146,1 indicate that the two individuals are siblings.
The SI calculated indicated that the DNA evidence suggests that the remains belong to two individuals that are 5.6 million times more likely to be siblings than to be two unrelated individuals.

The relationship of Tsar Nicholas II and Tsarina to the individuals found in the grave was also tested using the likelihood ratio given:
Ho/Null hypothesis: The DNA samples from bones 147 and 146.1 indicate that the two individuals are completely unrelated to the Romanov family.
Ha/Alternative hypothesis: The DNA samples from bones 147 and 146.1 belong to the children of Tsar and Tsarina.
LR calculated indicated that the DNA evidence suggests that:
Sample 147 is 4.36 trillion times more likely to be the daughter of Tsar and Tsarina than an unrelated individual.
Sample 146.1 is 80 trillion times more likely to be the son of Tsar and
Tsarina than an unrelated individual.

Criticisms regarding Gill et al’s., initial research were put to rest:
Doubts on authenticity of sequences: “only a 221-bp amplicon could be produced (possibly from endogenous degraded DNA template), but not a 400-bp nested product…. [the] results in
(Gill et al.) are not plausible’’ – Re-testing produced same results.
Doubts on point heteroplasmy: extremely rare/unknown in the past so critics argued that it was inaccurate analysis –
Same point heteroplasmy was found when bone samples from
Tsar Nicholas II were retested and also from his brother,
Grand Duke Georgij.
The bones found in the second grave were found to be related to those from the first grave: All seven members of the Romanov
Family had been murdered on July 17 th 1918 – No survivors.

Born in 1882 in Penza, Russia.
Studied Law at the Kharkov University.
Served as court investigator before the revolution and rose to the post of Major Case investigator in Penza.
After the revolution, he left Penza and disguised himself as a peasant.
Went to Siberia where he became Court Investigator.
Not soon after, he was assigned to the unsolved case of the
Romanovs.

Was summoned on February 5 th
1918 by the White Army
Commander and entrusted with the investigation.
Gathered physical evidence and took testimonies from servants, guards, teachers and other eye witnesses.
Evidence led him to conclude that the Romanovs had been murdered on the night of the
17 th .
At the time of the investigation, the Whites army was forced to the East but Sokolov continued his investigation.
The box in which the remains of the Imperial Family collected from the grave outside of
Ekaterinburg were place. This box was actually a Russian Army suitcase.

His investigation revealed disappointing news so many disbelieved.
Nikolai believed the Russian Public had a right to know so he published his findings from the investigation in a book called “ The Murder of
The Imperial Family”.

Began working with Alec Jeffery to use his research on DNA profiling to try and apply the concept to forensic casework.
Co-author of the paper that introduced forensic DNA testing.
His research led to the revolution in the area of forensic DNA profiling.
Approached by Russian authorities 6 years after his work woth Alec Jefferys to perform analysis on remains of
Tsar’s family – required scientific confirmation.
After his research on the Romanov’s confirmed their identity, he was involved in a subsequent investigation to disprove the claim of Anna
Anderson as being the Duchess
Anastasia.

Involved in providing evidence for many controversial cases.
Published more than a 100 peer reviewed papers in international scientific literature.
Currently:
Senior lecturer at the University of Strathclyde, UK .
Professor of Legal Medicine,
University of Oslo, Norway.
Chair of the DNA commission of the International Society of
Forensic Genetics.
Member on the editorial boards for Science Literature Journals.

DNA profiling is now an advanced and well understood concept that has many applications in the modern day:
Paternity tests
Proof of relatedness
Criminal Justice
Sex Testing
Identification

Single Nucleotide Polymorphisms (SNP's) : Errors made in the copying process during cell division lead to varied DNA sequences in regions called Single Nucleotide
Polymorphisms. These allow for DNA profiling of the Y chromosome.
Touch DNA : Just a few skin cells left behind at a crime scene (can even be as few as
7/8) can be duplicated and tagged at specific regions to produce a genetic portrait that allows identification of unknown individuals.
Collection of Buccal Cells for DNA Analysis : DNA from epithelial cells in saliva can be swapped and this allows quick screening of suspects because the samples can be analyzed quickly using techniques such as PCR.
SNP Based Ancestry Markers - Biographical Ancestry: Can be used to trace geographic roots of individuals and can also be used in identifying which of the four main continental group they originate from. This kind of information can be used by investigators to determine specific physical characteristics to look for in a suspect.
Combined DNA Index System (COIDS) is a national DNA database that allows public forensic labs to match their suspect DNA to persons whose DNA is on the system providing a fast and easy method to find a match

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