Transcript 1200 Paul Winter

Genetics of
Inherited Bleeding Disorders
Dr Paul Winter
N. Ireland Haemophilia Centre
Belfast City Hospital
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
How Blood Clots
• 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
• 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
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
Haemophilia Inheritance
XX
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
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%
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
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
DNA
 Linear molecule, very long
 Double Helix
 A, G, C, T (bases)
 DNA Sequence contains
information needed to make a
protein
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
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
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
Identifying F8 Mutations
1. Obtain blood sample from
male with Haemophilia
2. Extract DNA sample
3. Isolate his F8 Gene
(PCR)
4. Determine the DNA
sequence of his F8
Gene
5. Compare patient and
normal sequence
6. Look for a difference
(Mutation)
What is a Mutation?
 A change in the DNA sequence of a gene that causes an
inherited disease
 Two types of mutation:
1. Point mutations: a single base is changed
2. Gross mutation: a change involving a large piece of DNA
sequence (eg: deletion mutation)
Point Mutations
 Three types of Point Mutation cause Haemophilia:
1. Missense Mutations
2. Nonsense Mutations
3. Frameshift Mutations
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

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
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
Frameshift Mutations
3. Frameshift mutations:
 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
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
Mutations That Cause Haemophilia
are Very Diverse
Unique F8 Mutations (n = 2015)
Insertions
Large Deletions
Missense 983
Nonsense 208
Splice 158
Small Deletion 357
Large Deletion 255
Small Deletions
Insertion 146
Missense
Splice
Nonsense
Missense Mutation
F8 Exon 19
G to A point mutation at nucleotide 6089
Changes Serine 2030 to Asparagine
Serine 2030 is highly conserved
Sister of patient is heterozygous carrier of the same mutation
Carrier Haemophilia A
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
Patient’s sister is heterozygous carrier of same mutation
Carrier Severe Haemophilia A
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
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.
60% have
1 of 3
F8
mutations
Von Willebrand Disease
First Described by the Finnish
Physician, Dr Eric von Willebrand
in 1926
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 lifethreatening 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.
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)
Inheritance of VWD
VWF/VWF
VWF/ VWF
VWF/VWF
VWF/VWF
VWF/VWF
VWF/VWF
-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
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).
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
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
1.
2.
3.
4.
5.
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
VWF Protects Factor VIII
 VWF binds Factor VIII in plasma
 Protects FVIII from degradation
 No VWF binding: Factor VIII has short half life
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%)
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 plasmaVWF levels
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
Diagnosis of VWD
 Three Criteria:
 Laboratory tests indicating a deficiency of VWF
PLUS
 A personal bleeding history
 A family history of bleeding
Lab Tests for VWF
 VWF: Ag
- Measures the amount of VWF protein in plasma
- Immunoassay
 VWF: RiCoF
- Measures VWF activity in plasma
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
3 types of VWD exist
 Type 1 : Reduced amount of VWF in plasma
VWF:Ag andVWF: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 andVWF:RCo ~0%
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
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
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
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
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