Week 5 Coagulation Lecture 1 - Haemophilias & Rare Coagulation Disorders

Question 1

Haemophilia A & Haemophilia B

Answer 1

Haemophilia A is an X-linked hereditary disorder that is due to defective and/or deficient factor FVIII molecule
Haemophilia B is an X-linked hereditary disorder that is due to defective and/or deficient factor IX molecule

Question 2

Haemophilia A & Haemophilia B - Clinical Features

Answer 2

Lack of excessive bleeding from minor cuts and abrasion
- reflects ‘normal’ platelet function
Easy bruising
Soft tissue haematomas
Haemarthrosis and subsequent joint dysfunction
Prolonged and potentially fatal postoperative haemorrhage

Question 3

Types of Bleeds - Haematomas and Pseudotumours

Answer 3

Haematoma
- haemorrhage into connective tissue or muscle
- have a tendency to progressively enlarge and to dissect in all directions
Pseudotumour
- occur in 1-2% of patients with severe haemophilia
- usually present as a painless expanding mass growing slowly
- start as a simple cyst, following a bleed into a muscle or soft tissue
- as it increases in size it compresses and destroys the adjacent muscle, nerve, bone etc

Question 4

Types of Bleeds - Haemarthrosis

Answer 4

It is a bleed into the joints
Accounts for ~75% of bleeds
Haemophilic arthropathy is usually multiarticular
Ankles are commonly affected in children
Knees, elbows and ankles in adolescents and adults
Repeated bleeds leads to joint damage
Chronic toxic effects of blood and an inflamed synovium result in eroding into cartilage and subchondral bone, causing subarticular cyst formation

Question 5

Types of Bleeds - Neurologic Conditions

Answer 5

The most dangerous haemorrhagic event in haemophiliac patients
Accounts for 25% of the deaths in haemophiliacs
Bleeding may be subdural, epidural, subarachnoid, intracerebral or rarely intraspinal

Question 6

Classification of Haemophilia (Severe, Moderate, Mild)

Answer 6

Severe
- less than 1% FVIII:C/FIX
- bleeding occurs without known trauma
- haemarthrosis becomes frequent at about the time the patient begins to walk and chronic haemophilic arthropathy by early adulthood
- soft tissue haemorrhages dissect tissue planes and compromise vital organs
Moderate
- 1%-5% FVIII:C/FIX
- occasional haemarthrosis and haematomas, usually, but not always associated with trauma
- haemarthropathy is less disabling than in the severe patients
Mild
- 6%-30% FVIII:C/FIX
- infrequent bleeding episodes
- excessive haemorrhage following surgery or injury

Question 7

Laboratory Aspects - Haemophilia A

Answer 7

General:
- BT = normal
- PFA = normal
- [platelet] = normal
- platelet function = normal
Screening assays:
- PT = normal
- aPTT = abnormal (prolonged)
Specific assays:
- FVIII:C = abnormal
- aPTT 50:50 mix with normal plasma shows sustained correction
- FVIII.Ag distunguishes deficiency from dysfunctional proteins
- VWF.Ag = normal/increased
- VWF function = normal

Question 8

Laboratory Aspects - Haemophilia B

Answer 8

(Same as lab aspects of Haemophilia A)
General:
- BT = normal
- PFA = normal
- [platelet] = normal
- platelet function = normal
Screening assays:
- PT = normal
- aPTT = abnormal (prolonged)
Specific assays:
- FIX:C = abnormal
- aPTT 50:50 mix with normal plasma shows sustained correction
- FIX.Ag distinguishes deficiency from dysfunctional proteins
- VWF.Ag = normal
- VWF function = normal

Question 9

Haemophilia in Females

Answer 9

Rarely may affect females
Inherit two copies of affected F8 gene
- extremely rare - affected father and carrier mother
May also occur in female with other X-chromosomal abnormalities
- e.g. Turner syndrome (X0) or X mosaicism
Symptomatic carriers can occur:
- with factor levels as low as 20%
- due to extreme Lyonisation ‘normal’ X chromosome /F8 gene

Question 10

FVIII Protein Structure

Answer 10

The molecule is organised into six domains
They are arranged from the amino to the carboxy-terminus in the order (NH2)A1-A2-B-A3-C1-C2(COOH)
FVIII consist of a light chain comprising the A3-C1-C2 domains, and a heavy chain comprising the A1-A2 domains and a variable amount of the B domain
Thrombin and FXa both activate FVIII
After thrombin activation a portion of the B domain is removed
FVIII is a very labile and decays spontaneously in the absence of further proteolysis

Question 11

FVIII Complex

Answer 11

FVIII and vWF circulate in blood as a complex
Deficiency of FVIII (FVIII:C) leads to Haemophilia A
Deficiency/abnormality of VWF leads to Von Willebrand Disease
VWF ‘protects’ FVIII from proteolysis in circulation

Question 12

Haemophilia A Variant - Inversion Intron 22

Answer 12

~497 kb distal to 3’ end of the F8 gene
Interrupts F8 mRNA at intron 22
Makes up ~50% severe cases; 36% all cases

Question 13

Haemophilia A Variant - Inversion Intron 1

Answer 13

Int1h-1; 15.26 kb downstream of exon 1
Int1h-2; 125 kb upstream of exon 1
=> altered mRNA; short F8 mRNA, incl. exon 1 only
Uncommon; ~ 1%

Question 14

Haemophilia A Variant - Point Mutations

Answer 14

Affects all exons
‘Hot-spots’
- CpG sites (70)
- spontaneous deamination of methyl-cytosine
May produce missense or nonsense proteins
=> Range of clinical manifestations (mild, moderate, severe)
May affect splice sites

Question 15

Haemophilia A Variant - Insertions

Answer 15

Affect most exons
Largest number of mutations affecting exon 14 (54/109)
‘Hotspots’ within exon 14
- 9As; codons 1191-1194
- 8As; codons 1439-1441
- 6As; codons 1588-1590
None reported for exons 10, 15, 23, 26

Question 16

Haemophilia A Variant - Deletions

Answer 16

Size of deletion varies
Could be >1 base, 1 exon or entire gene
Affect most exons
- not exon 21
- exon 14; 114/266 small deletions

Question 17

Haemophilia A Variant - Splice Site Mutations

Answer 17

Affect splice sites
- => altered mRNA
Lack poly AAA tail
Lack stability
- => degraded
Altered sequence => altered protein structure
Most exons affected
- not exons 20, 26
- exon 5 most affected, 10 unique mutations

Question 18

Introduction to Rare Coagulation Disorders

Answer 18

Usually transmitted as autosomal recessive traits
They include inherited deficiencies of fibrinogen, FII, FV, FVII, FX, FXI, FXIII and combined FV and FVIII deficiencies

Question 19

FVII Deficiency

Answer 19

FVII deficiency is the most common RCD
Variable spectrum of bleeding problems
- epistaxis, gum bleeding, menorrhagia and other mucous membrane type bleeding are common with severe FVII deficiency (FVII:C <2 IU dL)
- bleeding into the central nervous system in between 15- 60% of cases
Cases often present shortly after birth and this presentation is associated with a high morbidity and mortality

Question 20

FVII Deficiency - Molecular Biology

Answer 20

The F7 gene is located at chromosome 13q34 & comprises 9 exons
Considerable proportion of mutations located on exon 8, which codes for the catalytic domain of FVII

Question 21

Type 1 and 2 Inherited Fibrinogen Disorders

Answer 21

Type I disorders affect the quantity of fibrinogen in circulation
- hypofibrinogenaemia is characterised by fibrinogen levels lower than 1.5 g/l, while afibrinogenaemia is characterised by the complete deficiency of fibrinogen
Type II disorders affect the quality of circulating fibrinogen:
- in dysfibrinogenaemia fibrinogen antigen levels are normal, while in hypofibrinogenaemia levels are reduced

Question 22

FI Deficiency

Answer 22

Bleeding due to afibrinogenaemia usually manifests in the neonatal period
Intracranial haemorrhage is the major cause of death
Both arterial and venous thromboembolic complications are observed
Hypofibrinogenaemia patients are usually asymptomatic with fibrinogen levels around 1.0 g/l

Question 23

FI Deficiency - Molecular Biology

Answer 23

Causative mutations can be divided into two main classes:
1. Mutations with no protein production at all
2. Mutations producing abnormal protein chains
In the majority of patients with afibrinogenaemia or hypofibrinogenaemia there is no evidence of intracellular accumulation of the mutant fibrinogen chain

Question 24

Factor V Deficiency

Answer 24

Homozygous individuals FV levels range from <1 to 10 U dL
Homozygous deficiency is associated with a moderately severe bleeding disorder
- typically presents in childhood with easy bruising and mucous membrane bleeding, in particular epistaxes and oral cavity bleeding
- gastrointestinal bleeding and haematuria may occur rarely
Molecular:
- mutation in the F5 gene

Question 25

Combined FV & FVIII Deficiency

Answer 25

Affected individuals have reduced plasma levels of both FV and FVIII
Mild clinical signs such as easy bruising and epistaxses are common
Circulating levels of FV and FVIII are usually sufficient to prevent more severe spontaneous bleeding episodes
However, bleeding is common following surgery, dental extraction and trauma
Prolonged PT and aPTT

Question 26

Combined FV & FVIII Deficiency - Molecular Biology

Answer 26

Single gene defect, rather than coinheritance of two
Caused by mutations in LMAN1 (ERGIC-53) and MCFD2 genes
Major role in intracellular trafficking of proteins including FV & FVIII from the endoplasmic reticulum to the Golgi intermediate compartment
Within hepatocytes FV and FVIII are synthesised normally but have defective passage of the factors through the cell and impaired release

Question 27

FX Deficiency

Answer 27

The prevalence of FX deficiency is greater amongst populations in which consanguineous marriage is common
Epistaxis is common
Menorrhagia occurs in ~ half women of reproductive age

Question 28

Moderate and Mild FX Deficiency - Clinical Features

Answer 28

Moderately affected patients (FX:C 1–5 IU dL) may bleed only after haemostatic challenge, e.g. trauma or surgery.
Mild FX deficiency (FX:C 6–10 IU dL) may be identified incidentally during routine screening or family studies