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Molecular coagulation andthrombophilia 213
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should be reassessed at least on an annual basis as the clinical
situation might change over time. Thus, the risks and benefits of an extended anticoagulation must be balanced with a
number of different considerations, including the patient’s
own preferences. However, despite new clinical data being
added all the time, there is no general consensus regarding
diagnosis, prophylaxis, and treatment of symptomatic
patients with VTE.
When the FV Leiden allele is present in homozygous
form, or heterozygosity is combined with a second genetic
defect, prophylactic treatment with heparin or oral anticoagulants is recommended in situations known to be associated with a high risk of thromboembolic complications, such
as surgery or pregnancy, even if the patient has never experienced any thrombosis or has no family history of such complications. For heterozygous asymptomatic carriers lacking a
family history of thrombosis, short- term prophylaxis has
been recommended in high- risk situations, but it remains to
be established whether prophylaxis should be given in all
situations associated with a risk of thrombosis.
Symptomatic heterozygous patients should be managed in
the same way as any other patient with thrombotic events
until more specific recommendations are established.
Current data suggests that there is no increase in thrombosis
recurrence risk in patients with heterozygosity for either factor V Leiden or the prothrombin gene mutation. Patients
with combined defects, and probably also patients with deficiency of antithrombin, protein C or protein S, may be at
increased risk of recurrence and should accordingly be given
long- term anticoagulation therapy beyond six months, even
after an isolated thromboembolic event. However, more data
are needed before these recommendations can be considered
generally applicable.
The potential benefits of general screening for APC resistance and/or the FV Leiden allele prior to thrombotic events
or in the presence of such circumstantial factors as oral contraceptive use, pregnancy, and surgery are obvious, but more
prospective data are needed, not least in terms of cost–benefit ratios before any general recommendations can be made.
Conclusions
Inherited APC resistance, caused by the Arg506Gln mutation in the FV gene (FV Leiden), is the most common genetic
risk factor for thrombosis identified to date. The mutated FV
has normal procoagulant properties, but the loss of the APC
cleavage site at position 506in FV results in impaired regulation of coagulation and a hypercoagulable state. The prevalence of FV Leiden in Caucasian populations varies between
2% and 15%. A genetic variant in the prothrombin gene
(G20210A) is another common prothrombotic risk factor,
with a prevalence of approximately 2% in the general popula-
tion. Other less common independent genetic risk factors
include abnormalities in the genes for AT, protein C, and
protein S. Families with thrombophilia present with variable
penetrance of thrombosis explained by different combinations of genetic defects and environmental risk factors.
Patients with combined genetic defects are at higher risk of
thrombosis than those with single gene defects. Thus, evaluation of patients with thrombosis must be performed in
order to fully estimate the risk for thrombosis in each case.
Further reading
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Chapter16
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The molecular basis ofhemophilia
Keith Gomez
Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, UK
Introduction: clinical features of hemophilia, 217
Inheritance of hemophilia, 218
Molecular basis of hemophilia A, 219
Molecular basis of hemophilia B, 221
Introduction: clinical features
ofhemophilia
The clinical severity (bleeding phenotype) is correlated with
the concentration of circulating factor VIII, or IX, in the
plasma, and severe hemophilia is defined by a clotting factor
concentration below 1 IU/dL (Table16.1). The hallmark of
severe hemophilia, in the absence of treatment, is recurrent
and spontaneous hemarthrosis. Typically, hinge joints such
as the knees, elbows, and ankles are affected, but bleeds may
also occur in the wrist or shoulder. Bleeding into the ball and
socket hip and shoulder joints is less frequent. The acutely
affected joint becomes swollen and warm, and held in a position of flexion (Figure16.1), with no external discoloration
or bruising around the joint. It is unusual for an infant to
suffer spontaneous hemarthroses in the first few months of
life, and the first joint to be affected tends to be the ankle as
the child learns to crawl. The first sign of hemarthrosis in an
infant will often be obvious discomfort and distress, accompanied by limping or reluctance to use a limb. Recurrent
bleeds into a joint, leading to synovitis and joint damage,
ultimately result in crippling arthritis (Figure16.2). Bleeding
into muscles is also a feature of severe hemophilia, but this is
usually a consequence of direct injury, albeit often minor
(Figure16.3). Bleeds into limb muscles are particularly dangerous because of the risk of compression of neighboring
structures manifesting as a compartment syndrome. Patients
with inhibitory antibodies to the deficient factor are particularly at risk in this regard, as bleeds may be more difficult
to control. Bleeds in the tongue can obstruct the airway, and
retroperitoneal bleeding within the iliopsoas muscle may
result in femoral nerve compression, causing paresthesia in
the anterior thigh, weakness, and wasting of leg muscles
(Figure 16.3). Bleeding from the gastrointestinal tract
(melena) and bleeding into the urinary tract (hematuria) may
Inhibitor formation: etiology and clinical implications, 222
Therapeutic applications of molecular biology to patient care, 224
Conclusions, 229
Further reading, 229
also occur. There is also a significant risk of intracranial
hemorrhage in severe hemophilia, which in the past was a
significant cause of mortality when treatment was not so
readily available. Higher levels of factor VIII, or IX, above
5
IU/dL are associated with a milder form of the disease, with
no spontaneous joint bleeds but a definite risk of bleeding
after even relatively minor injury or procedures (e.g. dental
extraction).
For decades, the only effective treatment was replacement
therapy with intravenous injection of coagulation factor concentrates (CFC). Preventive infusions of CFC are the gold
standard of care (prophylaxis therapy). Patients regularly
inject CFC intravenously (most commonly two to four times
a week) to prevent bleeds rather than just treating on demand
after bleeds or after an injury has occurred. The dose and
frequency of prophylaxis infusions are determined by the
half- life of the chosen CFC and increasingly after identifying
the pharmacokinetics (PK) of that CFC in the candidate
patient. Prophylaxis aims to maintain a measurable plasma
level of clotting factor activity above the patient’s baseline
(trough level) just prior to the next CFC prophylaxis dose.
Patients on prophylactic therapy experience few or even no
spontaneous bleeds, and thus progressive joint damage and
arthritis can be minimized or avoided. The quality of life of
patients on prophylaxis may be greatly enhanced, allowing
them to lead much more independent lives.
Approximately 30% of patients with severe hemophilia A
can be expected to develop inhibitory antibodies to factor
VIII CFC at some stage. In contrast, inhibitor development
in hemophilia B is very rare and encountered in less than 5%
of patients. The development of such antibodies poses considerable problems in treatment as these immunoglobulins
(IgG) are capable of rapidly inactivating infused factor VIII,
and furthermore, the antibody titer may rise dramatically
after a course of factor VIII (anamnesis). Very occasionally,
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
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217
218 Molecular Hematology
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Table16.1 The relation ofblood levels offactor VIII (or IX) tothe
severity ofhemorrhagic manifestations
Level
(IU/dL) Hemorrhagic manifestations
>40 No increase in bleeding symptoms
>5–40 Mild hemophilia. Bleeding typically occurs only after
significant injuries
1–5 Moderately severe hemophilia: Spectrum of bleed
phenotype– some with spontaneous bleeds and
early arthropathy, others experiencing bleeds
associated with injury, albeit often relatively minor
<1 Severe hemophilia with spontaneous and recurrent
bleeding into muscles and joints. Risk of intracranial
hemorrhage
Figure16.2 Radiograph of the knee of a patient with severe
hemophilic arthropathy. Joint replacement surgery was subsequently
carried out in this case.
Figure16.1 Acute hemarthrosis in severe hemophilia. This usually
arises in the absence of injury. The joints most frequently involved are
the knees, elbows, and ankles. The joint is swollen, warm, and tender,
but there is no external bruising or discoloration.
acquired hemophilia A may arise in a previously normal
individual due to the formation of autoantibodies directed
against factor VIII, and both males and females may be
affected. Hemarthrosis is unusual in acquired hemophilia,
and the principal manifestations are usually extensive superficial purpura and muscle bleeds. Acquired hemophilia arises
most often in the elderly, associated with underlying malignant or autoimmune diseases in approximately half of cases,
with a small, younger cohort associated with pregnancy.
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Figure16.3 Magnetic resonance imaging (MRI) scan showing
bilateral iliopsoas hemorrhage. This bleed was associated with a
complete but transient paralysis in both legs, as the femoral nerve is
located on the anterior surface of the muscle and may be compressed
in such cases.
Inheritance ofhemophilia
The genes for factors VIII and IX (F8 and F9, respectively)
are both located at the telomeric end of the X chromosome
and thus hemophilia is inherited as an X- linked recessive
condition. The daughters of affected males are obligate carriers, but the sons are normal. The phenotype remains constant within a family, so the daughter of a man with only
mild hemophilia may be reassured that she will not pass on
a severe form of the condition. However, up to a half of all
The molecular basis ofhemophilia 219
0
Cu
Removal of
signal (pre-) peptide
Binding to FIXa
50 100 150 200 kb
186 kbp
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cases of hemophilia arise in the absence of a previous family
history and are due to a new mutation. The most famous
example is that of Queen Victoria, who had a hemophilic
son (Leopold) and two daughters (Alice and Beatrice) who
turned out to be carriers. Forensic genetic testing on
exhumed remains of the assassinated Russian royal family
has since proven the royal hemophilia to be severe hemophilia B. There are instances of hemophilia affecting females
due to inheritance of the defective gene from both parents
and there are also case reports of hemophilia in females
withTurner syndrome (XO karyotype) and androgen insensitivity syndrome (XY karyotype). A proportion of females
carrying a hemophilia gene have factor activity levels, from
that corresponding gene, below recognized laboratory normal ranges through a process called skewed Lyonization.
These girls/women effectively have mild hemophilia and
should be identified and offered care commensurate
with their levels, as would be offered to boys/men with
equivalent factor levels. The remaining carrier women
havenormal laboratory range factor activity in their plasma.
Importantly, normal plasma factor activity can NOT be
used as a surrogate marker of whether a daughter of a carrier
mother is a carrier herself. Genetic testing will always be
required to clarify true carriership.
Molecular basis ofhemophilia A
Factor VIII is an essential cofactor for the activation of factor
X by activated factor IX (see Chapter17). Factor VIII must
itself undergo proteolytic cleavage at three distinct sites
through the action of thrombin before it becomes physiologically active. It circulates in plasma as a large glycoprotein
bound non- covalently to the larger protein, von Willebrand
factor (VWF). The factor VIII gene (F8) was first cloned in
1984. It is 186 kb in length and is situated on the long arm of
the X chromosome at Xq28 (Figure16.4). The factor VIII
gene consists of 26 exons, which range in size from 69 bp
(exon 5) to 3.1 kb (exon 14). The factor VIII mRNA is nearly
9 kb in size and encodes a mature protein of 2332 amino
acids. Approximately half of all cases of severe hemophilia
and all cases of mild and moderate hemophilia result from
89 11 12
1234 56 7
1
20 760393170920392192
PreA1
19 3551667
A1 BA2 A3 C1 C2
Cleavage by
Thrombin
Figure16.4 Translation of the F8 gene into the Factor VIII protein. The top panel shows the F8 gene with exons colored according to the
domains that are translated from them. Exon numbers are given above the gene map. The positions of the homologous sequences that are
involved in inversion events are shown (int1h- 1 and int22h- 1). The primary translated product is the FVIII precursor. Start and end amino acid
numbers for each domain are shown. Removal of the signal (pre- ) peptide generates the mature FVIII procofactor that circulates in plasma.
Cleavage by thrombin after the arginine residues shown in red activates the protein by removing the B and a3 domains.
10 13 14
A2 A3 C1 C2
72920382191 2351
a1
a1 a2
Arg372 Arg740 Arg1689
a2 a3
a1 a2
A1
B
A2 A3 C1 C2
2+
15 16 17 18 19 20 21 22 23 24 25
int22h-1int1h-1
2351 aa FVIII Precursor
Binding to VWF
a3
2332 aa
1383 aa
26
F8
Gene
Mature
FVIII procofactor
Activated FVIII (FVIIIa)
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220 Molecular Hematology
int22h-3 int22h-2int22h-1
Normal
T
Telomere
Centromere
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heterogeneous single nucleotide variations that occur
throughout the F8 gene.
By far, the commonest single genetic defect causing severe
hemophilia is an inversion in intron 22, which is encountered in as many as 40% of people with severe hemophilia A
in all ethnic groups. The inversion mechanism occurs as a
result of homologous recombination between nearly identical sequences in intron 22 and outside the gene (Figure16.5).
Homologous recombination is a key part of the evolutionary
process that promotes genetic diversity and trait development through generations. During meiosis, homologous
sequences recombine between both alleles of most genes, but
as males have only one X chromosome, the single F8 allele is
prone to recombination with extragenic sequences. This
results in a rearrangement that disrupts the gene. In sporadic
cases of hemophilia (those without a family history), the
rearrangement occurs in about 80% of cases during grandpaternal spermatogenesis. This means that 80% of mothers in
sporadic cases are themselves carriers through having inherited the hemophilia- causing variant. The inversion was previously identified through Southern blotting. This required
radioactive labelling of gene probes and for safety reasons is
no longer the preferred detection method. Most laboratories
now use either inverse polymerase chain reaction (PCR) or
long- range PCR for identification of this inversion. Inversions
in intron 1 of the F8 gene have also been identified as a
cause of severe hemophilia and this abnormality appears
tobe responsible for approximately 5% of all cases of severe
hemophilia. Since approximately half of all cases of severe
hemophilia are associated with these two inversions, it is
usual practice to screen samples from new cases for these
two abnormalities first.
Developments in molecular biology have permitted more
rapid identification of defects in hemophilia. Southern
blotting has been superseded by methods involving PCR
amplification of either patient DNA or material derived
from the reverse transcription of mRNA (RT- PCR).
Automated sequence analyzers, enabling sequencing of the
entire F8 gene, are now more affordable. Where not available, previous methods may still be relevant to identify
restricted areas of abnormal DNA in patients with hemophilia, which may then be targeted for specific attention.
These methods include amplification and mismatch detection (AMD), conformation- sensitive gel electrophoresis
(CSGE), denaturing gradient gel electrophoresis (DGGE),
high- resolution melting analysis (HRM), and pyrosequencing. Approximately 4% of cases of hemophilia are the consequence of gene deletions, which have been reported
throughout the gene and are very variable in size. As with
the intron 22inversion, most deletions are associated with a
severe clinical phenotype.
To date, over 3000 unique variants causing hemophilia A
have been identified of which about 2050 are single nucleotide or point variations (EAHAD F8 database at https://f8db.eahad.org/index.php accessed in June 2023) (Figure16.6).
Previously referred to as “mutations,” that term is no longer
recommended by the Human Genome Variation Society
because of the difficulty in establishing pathogenicity in
some cases. “Polymorphism” is no longer used for the same
reason and both terms have been replaced in clinical practice
ype 1 inversion
Figure16.5 The intron 22inversion. Mechanism of the intron 22inversion that causes 40% of cases of severe hemophilia A. The top panel
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shows the normal F8 gene with exons clustered and colored according to the domains they are translated into. Intron 22 contains a sequence in
red, int22h- 1, that has two homologous copies positioned some 400 kbp telomeric of the gene but orientated in the opposite direction. When
homologous recombination occurs during meiosis, the int22h- 1 sequence may recombine with one of the homologous copies: int22h- 2 or
int22h- 3. Because the sequences are orientated in opposite directions, this leads to inversion of the intervening sequence so that exons 1–22 of
F8 are now facing the wrong direction and separated from exons 23–26 by 400 kbp as shown in the lower panel. This leads to complete
disruption of the gene and abolition of transcription.
400 kbp
int22h-3 int22h-2 int22h-1
15–21 18–13 2–7
22 14 1
1 2–7 8–13 15–21 24–26
14
400 kbp
22
23
24–2623
<1%
Large indels
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7%
Small indels
16%
Missense/nonsense
47%
The molecular basis ofhemophilia 221
yield a GT mismatch that is inefficiently repaired. It is also of
interest that a missense mutation may be associated with
varying degrees of clinical severity. Thus, a C → T mutation
at nucleotide 1689within exon 14, resulting in the replacement of arginine by cysteine has been reported in association
with both severe and mild clinical phenotypes.
Complex
rearrangements
21%
2–5%
Figure16.6 Relative frequency of different variant types associated
with hemophilia A. Uniquely among monogenic disorders, complex
rearrangements in the F8 gene cause a fifth of all cases of hemophilia
and 45% of severe cases. Single nucleotide variants (shown in various
shades of gray) are still responsible for the majority of cases.
None
Splice
5%
Regulatory
by “variant” with a pathogenicity classification. The majority
of missense F8 variants result in non- severe forms of hemophilia A. A further 340lead to the creation of preliminary
peptide chain termination or premature stop codons and the
production of truncated factor VIII molecules devoid of any
functional activity. About 740 frameshift variations resulting
from insertions or small deletions have also been identified
as a cause of severe hemophilia. 936 unique copy number
variants (duplications, insertions, or a combination of both)
have also been identified in the F8 gene. Accurate identification of carriers of these variants requires specific techniques
such as multiplex ligation- dependent probe amplification
(MLPA). A full list of variants described in association with
hemophilia is outside the scope of this chapter, but additional information is provided in the Further reading section
at the EAHAD Coagulation Factor Variant Databases website (https://dbs.eahad.org). Knowledge of an individual’s F8
variant has relevance to predicting inhibitor risk: the more
severe the variant and resultant absence of translated protein,
increasing the risk. However, even missense variants resulting in single amino acid changes can still provoke an alloimmune response, resulting in clinically important antibody
formation (inhibitor).
Approximately 40% of all missense mutations arise at CG
dinucleotide sites, resulting in a change to TG or CA
sequences. It is generally believed that CG nucleotides represent genomic hotspots. Cytosine is predominantly methylated in human DNA, but this is relatively unstable and
5- methylcytosine is prone to spontaneous deamination to
Molecular basis ofhemophilia B
The factor IX gene (F9) is also located on the long arm of the
X chromosome at band Xq27, and is encoded by a stretch of
DNA spanning 33.5 kb that contains eight exons (Figure16.7).
The basic structure of the gene is similar in organization to
that of other serine proteases, such as protein C and coagulation factors VII and X, and it is likely that they all originated
in the distant past from a common ancestral gene by duplication. Factor IX mRNA comprises 2.8 kb and encodes a
mature protein of 415 amino acids. This is made up of a glutamic acid- rich sequence (Gla domain) and two epidermal
growth factor (EGF)- like domains separated from the serine
protease domain by an activation region.
Exons 1 and 2 encode the signal and pro- peptides
necessary for transport into the endoplasmic reticulum and
post- translational modification. Exons 2 and 3 encode the
Gla domain, which contains 12 glutamic acid residues that
undergo post- translational γ- carboxylation, which is necessary for binding of calcium. Once calcium is bound the Gla
domain can dock the protein onto negatively charged phospholipid surfaces, which is necessary for catalytic function.
Exons 4 and 5 encode the EGF domains which are involved
in factor VIII binding. Exon 6 encodes the activation peptide
that is cleaved off during the activation of factor IX by either
factor XI or a complex of tissue factor and factor VII.
Exons 7 and 8 encode the catalytic regions of factor IX,
which are responsible for the subsequent activation of factor
X in the coagulation cascade. The gene is controlled by a
promoter.
The F9 gene, cloned in 1982, is considerably smaller than
F8. The first defects identified in hemophilia B were gross
deletions, detected by Southern blotting. The EAHAD factor
IX database lists 1244 unique variants (https://f9org accessed in June 2023). Large gene insertion- deletions
account for 6% of all cases of hemophilia B with 32 unique
variants listed (Figure16.8).
There are 670 unique missense variants listed and 94nonsense. In contrast to missense F8 variants causing non- severe
hemophilia A, inhibitor formation in non- severe hemophilia
B is unheard of. The likely explanation is that factor IX shares
epitopes with other serine proteases which have a similar
quaternary structure. Our immune system is therefore less
likely to consider epitopes on exogenous factor IX protein as
foreign. Factor VIII, on the other hand, is not part of a
db.eahad.
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222 Molecular Hematology
0
Removal
of signal peptide
Phospholipid binding FVIIIa binding
10 20 30 40 kb
Gene
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1234 56 78
Exon
F9
1
Signal
Removal
of pro-peptide
Removal of
activation peptide
Figure16.7 Translation of the F9 gene into the FIX protein. Derivation of activated FIX from the F9 gene. The top panel shows the F9 gene with
exons colored according to the domains that are translated from them. Exon numbers are given above the gene. The primary translated product is
the FIX precursor. Start and end amino acid numbers for each domain are shown. Removal of the signal peptide generates the FIX procofactor.
The pro- peptide directs γ- carboxylation of glutamic acid residues in the Gla domain. Removal of the pro- peptide produces the mature protein that
circulates in plasma. Cleavage by FXIa after Arg191 and Arg226 removes the activation peptide leaving the active protease.
protein super family. Thereare some similarities to factor V,
but the sequence homology is relatively low. The unusually
high frequency of variants at CG dinucleotide sites in hemophilia B probably reflects the high number of CG dinucleotides at critical sites in the F9 gene.
Variants in the promoter region of the F9 gene (e.g. T
at −20 and G → A at −6) are relatively rare and account for
29 47 93 130 192 227
Pro Gla EGF1 EGF2 Catalytic domain
28 46 92 129191 461
γ–carboxylation
Pro Gla EGF1 EGF2 Activation
Gla EGF1 EGF2
Gla EGF1 EGF2
Activation
Activation
Cleavage by Factor XIa
226
factor IX levels in the one- stage activity assay. This variant
FIX Precursor 461 aa
Catalytic domain
FIX Procofactor 433 aa
Catalytic domain
Mature FIX Zymogen 415 aa
FX binding and cleavage
Catalytic domain
Activated FIX (FIXa) 380 aa
is now used in gene therapy for hemophilia B as a way
ofachieving the desired factor IX activity with lower doses
ofvector.
Full details of the many genetic abnormalities associated
→ A
with hemophilia B can be found at the websites listed at the
end of this chapter.
around 2% of all cases. However, they are of particular interest as they can give rise to the unique hemophilia B Leyden
phenotype, where the factor IX level rises significantly after
puberty with loss of the bleeding tendency (Figure 16.9).
Inhibitor formation: etiology
andclinical implications
Most of these variants are located in regions that contain
binding sequences for liver- enriched transcription factors,
which are influenced by androgen levels that rise during
puberty.
A gain- of- function F9 variant R338L, F9 Padua (substitution of leucine for arginine at position 338), results in juvenile thrombophilia rather than hemophilia B. This has been
shown to be because the variant molecular activates factor X
much faster than wild- type leading to much higher apparent
A proportion of patients with hemophilia will develop alloimmune immunoglobulins directed against infused factor
VIII (or IX) CFC. The individual’s immune system perceives
the infused, wild- type CFC as “foreign” because it has not
been tolerized through exposure to immunogenic epitopes
in the exogenous protein. This is potentially very serious, as
individuals become refractory to conventional doses of coagulation factor concentrates and bleeding can be very difficult
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