1. Genetics of Inherited Bleeding Disorders
Dr Paul Winter
N. Ireland Haemophilia Centre
Belfast City Hospital

2. Inherited Bleeding Disorders
A group of inherited diseases
Abnormal and prolonged bleeding
- Haemophilia: Coagulation defect,
deficiency of Factor VIII/IX
- VWD: Defect in platelet adhesion,
deficiency of Von Willebrand Factor
- Rare coagulation factor deficiencies

3. How Blood Clots Fibrin Forms a supporting network
Coagulation System makes
Fibrin
Forms a supporting network
around the platelet plug
Strengthens the clot
Seals the damaged area of the
blood vessel
Prevents further blood loss

4. Coagulation System is a
series of linked enzyme
reactions
Enzymes are proteins
called Clotting Factors
Each Factor given a
number
(Roman Numerals)
Factor VIII and Factor IX
are two of the most
important clotting factors

5. Inheritance of Haemophilia
Inheritance is X-Linked
- males affected
- females may be carriers
Haemophilia A: ~1/10,000 males
Haemophilia B: ~1/30,000 males

6. Haemophilia InheritanceXX XY XY XY XX XX
Mother is a Carrier:
50% of sons have Haemophilia
50% of daughters are Carriers
Important to identify female carriers for Genetic Counselling

7. Haemophilia A Lack of Factor VIII in plasma
Range of clinical severity:
Severe/moderate/mild
Severity related to Factor VIII activity (FVIII:C)
- Severe: 0-2%
- Moderate: 2-5%
- Mild: 5-30%

8. Haemophilia A Lack of Factor VIII in plasma is
caused by mutations in the gene
that makes the Factor VIII protein
F8 gene is located at the tip of the
X chromosome

9. What is a Gene?
A piece of DNA that contains all the information needed to make a protein
Human Beings have 35,000 genes.
Genes are spread out along the chromosomes
Each gene makes a different protein
Proteins do lots of important jobs in the cell

10. DNA Linear molecule, very long Double Helix A, G, C, T (bases)
DNA Sequence contains information needed to make a protein

11. Proteins Do all the jobs that need done in the cell
Proteins are strings of amino acids
30 different amino acids
Each amino acid encoded by 3 DNA bases (codon)
AGC = Arginine
TTC = Phenylalanine
GTC = Valine

12. F8 Gene F8 Gene is very large – 186,000 bases
Gene is split into Exons and Introns
The 26 exons encode the 2332 amino acid
sequence of the Factor VIII protein
The 26 exons cover 8,000 bases.
Haemophilia A is caused by a mutation of just one base

13. Mutation Screening
Males with Haemophilia are routinely screened to identify their F8 mutation
Two Purposes:
1. Confirm the diagnosis and the severity
2. Allows screening of related females who may be carriers and at risk of having an affected male child

14. Identifying F8 Mutations
1. Obtain blood sample from male with Haemophilia
2. Extract DNA sample
3. Isolate his F8 Gene (PCR)
5. Compare patient and normal sequence
6. Look for a difference (Mutation)
4. Determine the DNA
sequence of his F8
Gene

15. What is a Mutation?
A change in the DNA sequence of a gene that causes an inherited disease
Two types of mutation:
Point mutations: a single base is changed
Gross mutation: a change involving a large piece of DNA sequence (eg: deletion mutation)

16. Point Mutations Three types of Point Mutation cause Haemophilia:
1. Missense Mutations
2. Nonsense Mutations
3. Frameshift Mutations

17. Missense Mutations
Missense mutation: base change alters the amino acid encoded at that point
G>A mutation: AGC>AAC: Serine>Asparagine
C>T mutation: CGT>TGT: Arginine>Cysteine
Changing the amino acid alters the biological activity of the protein
Usually associated with mild Haemophilia

18. Nonsense Mutations 2. Nonsense Mutations
64 codons encode 30 amino acids
Three codons act as a signal to indicate the end of the gene during protein synthesis
STOP codons: TGA, TAA and TAG
A nonsense mutation is a base change that creates a new STOP codon
C>T: CGA>TGA: Arginine>STOP
C>A: TCA>TAA: Serine >STOP

19. Nonsense Mutations
Start Codon
Stop Codon
Nonsense mutations create a new STOP codon in the middle of the gene
Protein synthesis stops too early
A shortened protein is produced (inactive)
Usually causes severe Haemophilia

20. Frameshift Mutations ATT CAG GCA GAA ATA GTA TTT AGA GGG
Caused by the insertion or deletion of a base
Changes the order of the codons in the gene (reading frame)
ATT CAG GCA GAA ATA GTA TTT AGA GGG
I Q A E I V F R G
ATT CAG GTC AGA AAT AGT ATT TAG AGG G
I Q V R N V I STOP

21. Frameshift Mutations
A different set of amino acids is read after the inserted (or deleted) base
Biological activity of the protein is lost
Usually causes severe Haemophilia

22. Mutations That Cause Haemophilia are Very Diverse

23. Missense Mutation F8 Exon 19 G to A point mutation at nucleotide 6089
Changes Serine 2030 to Asparagine
Serine 2030 is highly conserved

24. Sister of patient is heterozygous carrier of the same mutation
Carrier Haemophilia A

25. Nonsense Mutation F8 Exon 24 C to T Point mutation at nucleotide 6682
Changes Arginine 2228 (CGA) to a STOP codon (TGA)
Premature termination of translation gives shortened protein (unstable)
Patient has Severe Haemophilia A

26. Patient’s sister is heterozygous carrier of same mutation
Carrier Severe Haemophilia A

27. Frameshift Mutation
Single base (A) deleted at nucleotide 3637 (8 A’s instead of 9)
Reading frame changes at codon 1213
Normal sequence: ATT CAG GAA GAA ATA GAA
I Q E E I E
Mutated sequence: TTC AGG AAG AAA TAG
F R K K STOP

28. Patient’s sister is heterozygous carrier of the same mutation.
Deletion of a single base on one allele causes sequences to be out of phase.

29. 60% have
1 of 3
F8 mutations

30. Von Willebrand Disease
First Described by the Finnish Physician, Dr Eric von Willebrand in 1926

31. In 1926, Erik von Willebrand published an article describing a bleeding disorder he had first observed in some members of a family from the Aland islands
The index case was a 5-year old girl who he documented as having a series of life-threatening bleeding episodes. At the age of 14 years, she subsequently bled to death during her fourth menstrual period.
Von Willebrand subsequently studied 66 members of the same family and found that 23 of them had symptoms of the same type.

32. Von Willebrand Disease
Most common IBD in Humans
Variable and mostly mild bleeding tendency
Inherited deficiency of Von Willebrand Factor (VWF)
Caused by VWF gene mutations (usually)

33. Inheritance of VWD Inheritance different from Haemophilia
Males and Females
affected
Autosomal Dominant: only
one defective gene needs to
be inherited for a person to
have the disease
50% of children of a parent
with VWD will have VWD
themselves
VWF/VWF
VWF/VWF
VWF/ VWF
VWF/VWF
VWF/VWF
VWF/VWF

34. Bleeding Symptoms in VWD
Distinct from haemophilia
Skin and mucous membranes affected:
easy bruising, epistaxis, menorrhagia,
excessive bleeding from minor wounds, dental extractions, surgery, childbirth, oral bleeding, GI bleeding
Unlike haemophilia, musculoskeletal bleeding
(haemarthroses, muscle haematomas) is rare
Serious bleeding only for severe forms (type 3 VWD).

35. Von Willebrand Factor VWF is a large plasma protein
Binding sites for FVIII, collagen and platelets
Important role in blood clotting
Mediates platelet adhesion and aggregation

36. VWF Multimers VWF synthesised in endothelial cells and megakaryocytes
Exists as a series of polymer
chains called Multimers
Multimer structure is important
for role of VWF in blood clotting
VWF stored in granules in
platelets and endothelial cells
VWF multimers released into
plasma in response to blood
vessel injury

37. Vessel Damage exposes subendothelium to blood
VWF binds to collagen
Gp1b binding site on VWF becomes exposed
VWF binds platelets via Gp1b
Platelets adhere to damaged area

38. VWF Protects Factor VIII
VWF binds Factor VIII in plasma
Protects FVIII from degradation
No VWF binding: Factor VIII has short half life

39. How Common is VWD? Plasma VWF ref range: 50-200 IU/mL
1% of the population have a level <50IU/mL
Clinically significant VWD only seen in 1:8,000 (0.125%)

40. VWF and Bleeding Risk
As VWF levels get lower, the risk of bleeding increases but relationship is not strong until the VWF level is very low
Mild bleeding is common in healthy population and may be due to factors other than VWF level
Diagnosis of VWD cannot be made only on the basis of low plasma VWF levels

41. Plasma VWF levels Vary Widely
Levels affected by genetic and acquired factors
Blood group is major genetic influence.
Group O 25% lower the Non-O
Acquired factors: Age, Exercise, Menstrual Cycle, Thyroid Function
Levels increase with age: 6%/decade after 40

42. Diagnosis of VWD Three Criteria:
Laboratory tests indicating a deficiency of VWF
PLUS
A personal bleeding history
A family history of bleeding

43. Lab Tests for VWF VWF: Ag
- Measures the amount of VWF protein in plasma
Immunoassay
VWF: RiCoF
Measures VWF activity in plasma

44. VWF:RiCoF Test Ristocetin is an obsolete antibiotic
Ristocetin binds to VWF and exposes Gp1b binding site
VWF can bind platelets spontaneously when ristocetin is present
Platelets + ristocetin + Patient plasma
Platelet aggregation measured (platelet aggregometer)
Aggregation is a measure of VWF activity

45. 3 types of VWD exist Type 1 : Reduced amount of VWF in plasma
VWF:Ag and VWF:RCo reduced in parallel
Type 2 VWD: Reduced plasma VWF activity
VWF:Ag > VWF:RCo (Ratio >2)
Type 3 VWD: rare, severe form
VWF:Ag and VWF:RCo ~0%

46. Type 2 VWD
Type 2A: Loss of the biggest (HMW) multimers, reduced platelet adhesion
Type 2B: VWF binds to platelets spontaneously
Type 2M: Loss of VWF ability to bind platelets
Type 2N: Defective Factor VIII binding

47. Type 1 VWD Reduced plasma VWF levels Two categories of patients:
1. VWF <20 IU/mL associated with:
VWF gene mutation
Significant bleeding
Strong family history of bleeding
75% of mutations are missense
Mutations cause secretion failure or rapid clearance from blood

48. Type 1 VWD 2. VWF levels of 30-50 IU/mL - Up to 1% of population
Less than 50% have a VWF gene mutation
Personal and family bleeding history are less convincing
VWF levels alone are not sufficient for a diagnosis of VWD
Levels should be seen as a modest risk factor for bleeding

49. Summary VWD is the most common IBD in humans
Results in a mostly mild bleeding tendency that affects males and females
Diagnosis requires an assessment of VWF levels in conjunction with a personal and family bleeding history
DNA testing is a useful tool to confirm diagnosis

50. Thank you for listening