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 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

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) 5. Compare patient and normal sequence 6. Look for a difference (Mutation) 4. Determine the DNA sequence of his F8 Gene

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)

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 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

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

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 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.

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 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

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

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 plasma VWF 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 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%

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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