Blood coagulation factor IX is an enzyme which is a serine protease which is closely related to trypsin. Factor IX is synthesised in the liver and its production is vitamin K dependant. From being synthesised in the liver, coagulation factor IX circulates the blood as a zymogen (inactive precursor), when it is activated and becomes part of a complex pathway (blood coagulation cascade). Coagulation Factor IX is also known as F9 or Plasma Thromboplastic Component, if the gene is defected it can cause haemophilia B (Christmas disease). Factor IX is found on the long arm of the X chromosome as seen in the Diagram 1, the descriptive make up of FIX can be seen in a gene map (diagram 2).
Factor IX is roughly 34 kb long, it contains eight exons and its DNA transcript is 2803 bases in length. As Factor IX and its disorder are X linked, naturally it is males who have the disorder as males only have one X chromosome. Therefore Factor IX is carried by females and its disorder is recessive, however, in rare cases Haemophilia B has been found in females that have more than one defective factor gene.
Factor IX and the Coagulation Cascade
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Blood Coagulation is the process where strands of fibrin (fibrous protein) form a mesh that binds to various components in the blood to form a blood clot. Coagulation involves many activation steps of a number of factors that are normally present in the blood in an inactive form (table 1). The process is a cascade of reactions that has a domino effect, one activated factor activates another:
Zymogen (inactive factor in blood)
Activated clotting factor
Activated clotting factor
The blood coagulation cascade has both intrinsic and extrinsic pathways; these pathways are needed together for normal hemostasis. Factor IX plays a role in the intrinsic pathway. The intrinsic pathway is activated when a blood vessel is damaged and blood comes into contact with collagen, at this stage damaged platelets release phospholipids. This starts off the cascade to which some of the factors shown in table 1 are activated (diagram3).
As shown in diagram 3, Factor IX is activated by Factor XIa and Ca2+, once Factor IX is activated it then acts together with the platelet phospholipids and Factor VIII to activate Factor X. When Factor X is activated by both the intrinsic and extrinsic pathways it will follow down the final common path where fibrinogen (soluble) is converted into fibrin (insoluble) is produced therefore stopping the bleeding and forming a blood clot.
Mutation of Factor IX
Factor IX and its mutations have been thoroughly researched and therefore many mutations of this gene have been discovered. However I will only be discussing the effect of a single point mutation within the gene DNA. A single point mutation is when a single base pair is substituted, for example, in Factor IX there is a single point mutation where adenine (A) is substituted for a guanine (G). Therefore the original codon taken from the DNA sequence changes so that GAG, becomes GGG. This may not seem such a vast change, however, if this does occur the codons for amino acids change, in this instance a Glutamic acid is replaced with a Glycine. By doing this many things could occur. Glutamic acid is a large amino acid with an acidic side chain that is a proton donor, at physiological pH Glutamic acid is negatively charged. On the other hand Glycine is a small amino acid with a non polar side chain that does not give off protons or participate in bonding.
Therefore if this particular section of Factor IX was at the binding site of the enzyme then this exchange could potentially denature the binding site as there would be no charge for the substrate to attach to. This could cause many alterations in the genes activation. As Factor IX is activated by Factor XIa this could be potentially stopped if the binding site is denatured. Failure to activate Factor IX can deactivate the successive reactions (diagram 3), therefore Factor X is not activated from the intrinsic pathway and cannot continue down the final common path. This can cause only Factor X entering the final phase from the extrinsic pathway, this is not a sufficient amount of Factor X to have effective amounts of fibrin being formed. Reduced amounts of fibrin being formed will slow down the process of clots being formed and can take an individual an extended amount of time to stop bleeding from vascular damage. This condition is known as haemophilia B or commonly known as Christmas disease.
Polymerase Chain Reaction (PCR)
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PCR is a process used to amplify a particular DNA sequence. This process allows for a small sample of a DNA to be synthesised into millions of copies of the original specific nucleotide sequence within a couple of hours. For the reaction you require the original sample of DNA and its known primers. Primers are oligonucleotides that are single-stranded sequences of DNA that correspond to the sequences of the gene to be copied. Two primers are required for the reaction, the sense primer and the antisense primer. The primers used for Factor IX replication are; sense primer 5'-AGTCATCGCTATTACCATGG-3' and the antisense primer 5'-GATTTCAAAGTGGTAAGTCC-3'. Firstly, a solution is made with the original sample of the DNA, its primers, DNA polymerase and dioxyribonucleoside triphosphates (dNTP) a reaction buffer to maintain the pH, Magnesium Sulphate (MgSO4) and nuclease free H2O. This solution is then heated to 94oC for 20 seconds; this is done so that the original sample of DNA is denatured. By doing this, the strand of DNA is “unzipped” allowing the primers to attach to their corresponding bases. Then the mixture is put through the following procedure for 35 cycles to amplify and anneal;
- 72oC for 3 minutes,
- 58oC for 2 minutes,
- 72oC for 4 minutes.
After 35 cycles the solution is heated to 72oC for a further 7 minutes for extension of the sample. Once this has been achieved there is two different ways to analyze the amplified DNA sequence,