Hemophilia Genotype Analysis Identifies the Cause of the “Royal Disease”

Article & Background

Hemophilia is a genetic disorder that impairs the body's ability to make blood clots- a process needed to stop bleeding.
The disease played an significant role in European history because it affected the children of Queen Victoria. It was called the "Royal Disease" because it spread throughout the royal families of Europe.
Figure S1
The "Royal Disease" is an example of X-linked recessive inheritance.

Two Candidates for the Gene Mutation

F8 Gene

  • Located on the X chromosome
  • Encompasses ~187 kb pairs
  • Encodes for coagulation factor VIII
  • Mutation in this gene is responsible for Hemophilia A, which is the more common of the two type
  • (http://www.thinglink.com/scene/632969451821596674)

F9 Gene

  • Located on the X chromosome
  • Encompasses ~33 kb pairs
  • Encodes for a vitamin K-dependent coagulation factor IX
  • Mutation in this gene is responsible for Hemophilia B, also known as “Christmas disease”.
  • (http://wiki.ggc.usg.edu/wiki/Haemophilia_B)

Experimental Design

DNA Extraction

  • mtDNA and STR analysis was used to extract segments showing no significant contamination by foreign human DNA
  • mtDNA (mitochondrial DNA) analysis: shows you your basic maternal ancestry reaching back hundreds of thousands of years. It includes testing to determine your basic mitochondrial DNA haplogroup placement.
  • STR (short tandem repeat) analysis: used to compare specific loci on DNA from two or more samples

Multiplex PCR Amplification

Figure S2A
  • Because of the poor and limited DNA quality, short, overlapping fragments were used, then organized into sets
  • For F8 gene: 14 amplification sets, 7-30 primers each set
  • For F9 gene: 3 amplification sets, 7-30 primers each set
  • Two negative controls, PCR control and blank DNA control
  • Two sequencing strategies were used: one being Massively Parallel Illumina Ga sequencing (which we will look at further), and the other ABI Sanger sequencing on a 96-capillary 3730x

Massively Parallel Illumina GA Sequencing

Source: http://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf

Mutation in the F9 Gene

  • mtDNA genome sequencing confirmed the N7 samples were from Alexandra
  • There was only one explicit mutation identified in these secondary amplicon reads, which was an A to G mutation located on a intron, three base pairs upstream from exon 4.

Exon Trap Experiment

Figure S4A
  • Exon 4 on the F9 gene was cut out and cloned into an exon trap vector
  • Vector was placed into living cells to undergo splicing and transcription
  • Resulting RNA was isolated, quantified, and reverse transcribed into cDNA
  • cDNA was sequenced using MPS

SNP Analysis

Researchers looked into population SNP databases for:

  • non-affected individuals
  • unrelated, unaffected individuals of Caucasian ancestry
  • Hemophilia B patients

Discussion

  • This study has lifted more of the fog and mystery surrounding the Romanov family
  • Results showed that Prince Alexi was hemizygous for a mutation on the F9 gene
  • This mutation was an A to G transition, resulting in an AG sequence that created a premature, incorrect splice acceptor site
  • This incorrect gene splicing created a premature stop codon, and therefore a truncated protein, Factor IX, which is responsible for blood coagulation, and if deficient leads to Hemophilia B, or, Christmas disease.

Bibliography

Credits:

Created with images by qimono - "blood cells red" • irinaraquel - "John Partridge - Queen Victoria" • NIAID - "Ebola Virus From Mali Blood Sample" • Andrew Mason - "Blood Cells"

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