Learning objectives
- To be able to synthesize a coherent and succinct set of key information regarding the genetic aspects of a monogenic disorder. (haemophilia A)
- To able to identify the key aspects of procedures used to synthesize and purify recombinant protein to understand how these may differ from the original protein
- To be able to identify the key areas of current or likely future research in this area particularly in the areas of diagnostic prognostic and treatment advances.
What is Haemophilia A?
Haemophilia A is lack of the protein factor 8, which is essential for the coagulation cascade. WHile it is usually genetic (X-Linked recessive) there are rare cases of acquired haemophilia. (give factor & as it can bypass 8)
Levels of haemophilia A mutations
Mutation can result in these levels of activity
- Severe (<1% normal activity)
- moderate (1-5% normal activity)
- or mild (5-30% normal activity)
Genetics of haemophilia A
This is an x linked recessive gene. this means mostly males are effected but female carriers can too due to x inactivation. The FVIII gene is large and near the telomeres. In 90% of mild-moderate cases point mutations or small deletions are at fault. 10% are due to splice site mutation or promotor mutations
40% of severe cases are due to a specific inversion event. there is a repeat element the gene which can cause the chromosome to self bind which, when replicated leads to a gene inversion
A bit about the factor 8 protein
This protein is made in the liver and is cleaved by thrombin into its active form. multiple glycosylations are needed and therefore it is processed through the ER.
Mutations in factor 8 gene
Mutations in the factor 8 gene can have different effects on the protein. Some are listed:
- change thrombin cleavage and prevent activation
- change binding to vWF
- Alter secretion
- Alter interaction with f 9 or 10
- destabilise protein folding
Treatment
Usually 3 products were used for haemophilia a
- plasma
- factor 8 concentrates from plasma
- recombinant factor 8
Factor 8 concentrates
Factor VIII concentrates from plamsa the aim is to increase levels to 30-50% during bleeding purified in a complex with vWf and concetrated from larfe amounts of pooled donated blood using biochemical techniques. life expectation rises to normal
however…
HIV and prions were a problem. so we have recombinant factor 8
Recombinant factor 8
Grown in chinese hamster ovary cells. this allows the protein to be glycosylated
2 main recombinant are advate and adynovate advate is 13-18 h half life adynovate has 1.5x half life
Factor 8 inhibition via immune system
The patients however can become intolerant to factor 8. this is due to the fact that it is a foreign antibody etc
Emicizumab
Factor 8 works via binding together factor 10 and 9a to make Xa. Emicizumab had one arm that binds to 9a and one that binds to 10 a and does this with factor 8