Genetic Risk Factors and Inhibitor Development

in Hemophilia: What Is Known and Searching for

the Unknown
Maurizio Margaglione, MD1 Mariano Intrieri, MD2

1 Medical Genetics, Department of Clinical and Experimental Address for correspondence Maurizio Margaglione, MD, Medical
Medicine, University of Foggia, Foggia, Italy Genetics, Department of Clinical and Experimental Medicine,
2 Department of Medicine and Health Sciences “V. Tiberio,” University University of Foggia, Viale Pinto, Foggia 71122, Italy
of Molise, Campobasso, Italy (e-mail: m.margaglione@unifg.it).

Semin Thromb Hemost

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Abstract Over the past few decades, important knowledge on why inhibitors develop and better
information about significant risk factors have become available. A series of both
genetic and nongenetic factors are recognized and clinical score systems were
proposed to quantify the risk for each patient. In addition, modulation of the
immunological response was acknowledged to play a pivotal role in the occurrence
of inhibitors. However, with the exception of mutation testing in severe hemophilia B
patients, no single risk factor or clinical score is currently utilized in clinical practice.
Keywords “Omics” technologies are large-scale hypothesis-generating approaches, which pro-
► hemophilia vide the tools to study issues contributing to a complex and multifactorial phenom-
► genetics enon, such as inhibitor development. Newer cutting edge technologies may enable a
► inhibitors more accurate estimation of the personal risk profile and provide a reliable tool to
► variants accurately measure the risk periodically, thereby enabling strategies to foresee and
► genomics prevent inhibitor formation.

Adequate therapy for both hemophilia A (HA) and B (HB) has
been available for the past four decades. Therapy typically
consists of coagulation-factor replacement administered
every 2 to 3 days, either prophylactically (i.e., to prevent
bleeding episodes) or episodically (on demand when bleed-
ing occurs). Such replacement treatment has led to consider-
able improvements in the life expectancy and physical status
of patients.1 At present, the quality of their life is similar to
that of the general population and this goal can be achieved
with adequate replacement therapy, using either recombi-
nant products or plasma-derived clotting factors that
undergo extensive purification and viral inactivation.
The development of alloantibodies (inhibitors) against
therapeutically administered factor VIII (FVIII) or factor IX
(FIX) impairs the effectiveness of treatment, and is the most
serious and challenging complication in treating patients
with hemophilia. Inhibitors compromise treatment with
plasma-derived clotting factors or recombinant products
by neutralizing FVIII and FIX activity, and will preclude
such treatment in the case of high inhibitor levels. During
their lifetime, the incidence of inhibitors is
20 to 30% in
severe HA and 3 to 5% in HB patients.2 Because of the higher
levels of morbidity and mortality and a worse quality of life,
attempts to restore the efficacy of replacement therapy are
mandatory. Why most patients are tolerant to the “foreign”
protein being administered, whereas some patients develop
neutralizing antibodies, is far from clear and it is a matter of
considerable discussion.
The clinical and economic burden of the management of
patients with persistent inhibitors fostered substantial

Issue Theme Alloantibodies and Copyright © by Thieme Medical DOI https://doi.org/

Congenital Bleeding Disorders: New Publishers, Inc., 333 Seventh Avenue, 10.1055/s-0038-1660816.
Insights in the Pathogenesis and New York, NY 10001, USA. ISSN 0094-6176.
Management; Guest Editors: Antonio Tel: +1(212) 584-4662.
Coppola, MD, Massimo Franchini, MD,
Cristina Santoro, MD, PhD, and Annarita
Tagliaferri, MD.
Inhibitor Development as a Multifactorial Event
research effort to elucidate the etiology of inhibitors and
several risk factors for their occurrence have been identified.
This effort allowed for increased knowledge on the genetic
and environmental determinants of the risk of inhibitor
development and the identification of predictors of devel-
oping inhibitors.
Notwithstanding, the identification of risk factors
(►Table 1) remains crucial to optimize candidate selection
to individualized treatment regimens or modification of
immunological factors according to the predicted risk and
might be of help for the detection of strategies for the
prevention of inhibitors and a decrease in the total incidence
of inhibitors among hemophilia patients.
Nonmodifiable Risk Factors
Genotype
HA is an X-linked bleeding disorder caused by heterogeneous
mutations in FVIII gene (F8). The incidence of HA is estimated
to be between 1:5,000 and 1:10,000 in men.3 F8 maps to the
distal end of the long arm of the X-chromosome (Xq28) and
spans 186 kb of genomic DNA. It consists of 26 exons and
encodes a mature protein of 2,332 amino acids.4 Over the last
few decades, rapidly increasing numbers of causative gene
alterations have been described in different ethnic groups. At
present, more than 900 mutations within the F8 coding and
untranslated regions have been identified and listed in the F8
variant database (URL: http://www.factorviii-db.org). These
findings support the concept that HA has a very high muta-
tional heterogeneity and represents a challenge for the
provision of genetic services because carrier and prenatal
diagnosis cannot be based on the screening of a limited
number of common mutations.
The type of F8 mutation is one of the most important risk
factors predisposing to inhibitor development in HA.5 In HA
patients, the majority of the inhibitors are directed against
Table 1 Established and proposed risk factors for inhibitor development
Disease Risk factor
HA Nonmodifiable F8 gene mutation
Family history
Ancestry
HLA alleles
IL-10 common alleles
TNF-α common alleles
CTLA4 common alleles
ABO blood group
Modifiable Traumatic injury or surgical challenges
High dosage and/or prolonged treatment at the beginning of the therapy
Primary prophylaxis vs. on-demand treatment
Recombinant products vs. plasma-derived high purity concentrates
HB Nonmodifiable F9 gene mutation
Abbreviations: HA, hemophilia A; HB, hemophilia B.
Seminars in Thrombosis & Hemostasis
Margaglione, Intrieri
the A2 and C2 domains.6 The C2 domain contains binding
sites to von Willebrand factor and phosphatidylserine on
activated platelets. Specific F8 variants, such as Arg593Cys in
the A2 and Trp2229Cys in the C2 domain, are highly related to
inhibitor development. On the other hand, replacement
therapy in patients completely lacking FVIII may lead to
immunization with the “foreign” protein and cause the
development of autoantibodies. Indeed, mutations predict-
ing a null allele develop an inhibitor more frequently (24–
71%), compared with missense mutations (5%). These find-
ings support the hypothesis that trace amounts of FVIII
protein, albeit nonfunctional, which are synthesized are
sufficient to induce immune tolerance in most HA patients.
In addition, mutations at specific regions of F8 (hot spots,
exons coding for the heavy chain) are associated with a lower
risk of developing inhibitors. Indeed, the risk of inhibitor
formation was similar in patients carrying the inversion of
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the intron 22 when compared with those with intron 1
inversion (odds ratio [OR] 0.9; 95% confidence interval
[CI]: 0.6–1.5) and splice site mutations (OR: 1.0; 95% CI:
0.6–1.5), but significantly higher in those with large dele-
tions and nonsense mutations (OR: 3.6; 95% CI: 2.3–5.7), and
lower in HA patients carrying small deletions/insertions and
missense mutations (OR: 0.5; 95% CI: 0.4–0.6).5
It is well known that the degree of severity of the disease
represents an important factor for the occurrence of inhi-
bitor. At variance with severe patients, the inhibitor risk is 5
to 10% in mild to moderate HA.7,8 Of note, severe and
nonsevere HAs display significant differences in prevalence
of type of causative mutations.9 Severe and nonsevere HAs
are also likely to be associated with different timing in risk of
inhibitor development.8
Similar to HA, HB is an X-linked bleeding disorder caused
by heterogeneous mutations in the FIX gene (F9). The inci-
dence of HB is estimated to be
1:25,000 in men.3 F9 maps to
the distal end of the long arm of the X-chromosome (Xq27),
Level of evidence
Well established
Well established
Established
Weak
Some evidence
Some evidence
Some evidence
Weak
Some evidence
Some evidence
Some evidence
Evidence
Well established
 Inhibitor Development as a Multifactorial Event
very close to F8, and spans 34 kb of genomic DNA. F9 consists
of 8 exons and encodes a mature protein of 461 amino acids.4
At variance with patients with HA, those with HB can
experience anaphylactic reactions to FIX concentrates at
the time of inhibitor development.10 The smaller molecular
size of FIX compared with FVIII, allowing for a rapid diffusion
into extravascular spaces, and the higher amounts of exo-
genous protein given to HB patients, due to physiologically
higher plasma levels of FIX (5 µg/mL) than FVIII (0.1 µg/mL)
may, in part, explain reasons for anaphylactic response.11
Although there have been occasional reports of large dele-
tions, HB is usually associated with hundreds of different
missense, nonsense, frameshifts, and splicing mutations.12
These gene variants frequently result in the production of a
defective, nonfunctioning, but immunologically detectable
FIX in the plasma of HB patients (cross-reacting material
positive [CRMþ]). Individuals with large gene deletions,
frameshift, and nonsense mutations are usually CRM and
are most susceptible to the development of inhibitors against
FIX, accounting approximately for 70% of cases, while mis-
sense mutations, which constitute the majority of genotypes
in HB, are at very low risk of inhibitor formation.13–15 The
increased risk of inhibitors in patients carrying large gene
deletions, frameshift, and nonsense mutations has prompted
recommending mutation testing in severe HB patients to
identify who are at risk for inhibitor development and
anaphylactic reactions.16 In addition, entity of risk in pre-
viously untreated severe HB patients is poorly known, as
most studies do not provide specific information and that
factors other than genotype have not been studied in such
patients.17,18 Thus, information reported in the following
paragraphs refer to HA.
Family History
Family history is also a risk factor associated with the
development of inhibitors.19–21 In a study, in which 249
families with severe HA containing two or more siblings
were evaluated, a high concordance between siblings (78.3%)
and a significant relative risk (3.2; 95% CI: 2.1–4.9) for the
development of an inhibitor was found.20 Noteworthy,
although higher than that observed in non-twin siblings,
concordance was not absolute (90%) even in monozygotic
twins.22 On the other hand, concordance in families with
inhibitors was 42%, and 72% of these inhibitors had the same
anamnestic (high-responding) features.21 Thus, the lack of a
complete concordance between siblings (all or none with a
history of inhibitors) shows that this genetic susceptibility
could not be predicted by the F8 mutation itself.
Ancestry
It is well known that HA patients of African or Hispanic
heritage have an increased risk of inhibitor develop-
ment.20,23–26 This association was recently confirmed in a
study that controlled for the F8 genotype.27 Differences in
amino acids between currently available replacement pro-
ducts (which are all from strains harboring F8 H1 and H2
haplotypes), and patients of African/Hispanic ancestry (more
commonly carrying H3 or H4 haplotypes), have been sug-
Margaglione, Intrieri
gested to explain this association.28 However, at variance
with this hypothesis, patients of African/Hispanic ancestry
with the H1 haplotype still had significantly higher inhibitor
frequencies than Caucasians, suggesting that the mechan-
isms accounting for these ancestry differences remain
unclear.29
Genes Encoding Immune-Regulatory Cytokines
As demonstrated in autoimmune disease, defects in genes
leading to an altered function of cytokines or their receptors
may cause an imbalance of immune response. Recently,
Astermark et al found a strong association between poly-
morphisms in the promoter regions of the interleukin-10
(IL10), tissue necrosis factor (TNF)-α, and the cytotoxic T-
lymphocyte antigen 4 (CTLA4) genes and the development of
FVIII inhibitors.30–32 These genes are known to be involved in
the regulation of antibody responses and could, therefore, be
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of major importance in the development of FVIII inhibitors.
The CTLA4 protein is a surface molecule expressed on
activated T cells and plays a critical role as a negative
regulator of T cell activation. Some polymorphisms of the
CTLA4 gene (promoter 318 C/T and exon 1 þ 49 A/G) are
associated with different levels of expression of this mole-
cule on the cell surface. A significant protective association
between the inhibitor development and the 318 T allele,
but not the þ49 A/G polymorphism, has been found.33
IL-10 is an important cytokine with a broad spectrum of
biological activities including immunosuppressive, anti-
inflammatory, and B cell stimulating properties. An
increased inhibitor risk has been shown in patients with a
microsatellite polymorphism in the promoter of the IL-10
gene compared with noncarriers.30
TNF-α is a proinflammatory cytokine that plays a key role
in the pathogenesis of many infectious and inflammatory
diseases. Many studies have investigated the TNF-α poly-
morphisms in autoimmune diseases in which the dysregula-
tion of TNF-α production might have played a role. In
particular, a SNP in the promoter region, 308 A/G, is
relevant: the TNF-α 308 A allele has been found to correlate
with enhanced spontaneous and stimulated TNF-α produc-
tion. The polymorphism is associated with antibody forma-
tion in patients with myasthenia gravis.24 A similar
association with the occurrence of inhibitors has been shown
in a subgroup of patients with severe HA enrolled in the
Mälmo International Brother Study (MIBS).33 The potential
role of these polymorphic markers is further supported by
their distinct distribution in different populations.33 Finally,
significant associations with the risk for inhibitor formation
were found with haplotypes at various loci of the IL-10, IL-1,
IL-2, and IL-12 genes.34 However, a more comprehensive
genetic approach enlisting a large number of brother pairs
discordant for inhibitor status did not confirm a pivotal effect
of CTLA4, IL-10, and TNF-α gene polymorphisms.35
Other Loci
The ABO blood group has been suggested to significantly
modulate the appearance of inhibitors. The relative risk in O
versus non-O blood group was 0.55 (95% CI: 0.33–0.92) and
Seminars in Thrombosis & Hemostasis
Inhibitor Development as a Multifactorial Event Margaglione, Intrieri
0.40 (95% CI: 0.21–0.77) for any and for high-titer inhibitors,
respectively.36 Because the glycosyltransferase activity,
which is encoded by the ABO gene, also significantly affects
FVIII and its carrier, von Willebrand factor, the differences in
posttranslational glycosylation may provide an intriguing
explanation for the differential risk. The HLA locus has been
suggested as another important genetic determinant capable
of influencing the occurrence of inhibitors in HA.33,37 HLA
class II molecules mediate the processing of antigenic extra-
cellular proteins, such as the exogenous FVIII administered to
HA patients. Findings from a series of studies showed a
significant association between the HLA system, in particular
HLA class II molecules, and the development of inhibitors.
However, the strength of this association varies throughout
the world and depends on the genetic background of the
different groups of population investigated.38
Final Considerations
Although it is well known that a genetic predisposition to
inhibitor development to FVIII/FIX proteins exists, it is also
clear that the classification into high-risk and low-risk
mutations is likely an oversimplification. From a clinical
point of view, to better define the individual risk for the
occurrence of inhibitors in newly diagnosed patients starting
replacement treatment, it would be helpful to also consider
the inhibitor family history and ancestry, which may both
include other genetic determinants.
Possible Modifiable Risk Factors
Treatment Related
Although the large proportion of patients who develop
inhibitors have severe disease, inhibitors can also occur in
patients with mild and moderate hemophilia. Likewise,
inhibitors are most common among previously untreated
patients and the risk decreases as the number of exposure
days rises. Inhibitors can also occur in patients who have
been already exposed for more than 50 days. However, in this
clinical setting, the overall rate of inhibitors is low.39 More-
over, inhibitor development often occurs in association with
traumatic injury or surgical challenges that result in signifi-
cant and prolonged exposures to replacement factor con-
centrates. The presentation to the immune system of the
exogenous FVIII alone may not be sufficient for initiating an
immune response. The intensity of the FVIII exposure has
been suggested to be a risk factor for inhibitor formation
because significant cell injury or inflammation leads to
immunologic “danger signals” that stimulate antigen-pre-
senting cells and upregulate an immunologic response
against exogenous FVIII, which could promote inhibitor
development.40–42 Several studies have shown a possible
link between intensity (high dosage and/or prolonged treat-
ment) of early treatment and increased risk of inhibitor
development.25,43–45 This prompted the concept that pri-
mary prophylaxis has a significantly lower inhibitor rate as
compared with patients treated on demand. The hypothesis
was confirmed in a recent patient-level meta-analysis.46
However, prophylaxis seems to be associated with a
Seminars in Thrombosis & Hemostasis
decreased overall inhibitor incidence after 20 days of expo-
sure to exogenous FVIII. The association was more evident in
severe HA patients with low-risk than in those with high-risk
F8 genotypes.47
Type of Factor Concentrate
Purified FVIII products were available in the 1970s, whereas
recombinant FVIII products became available in the early
1990s. The retrospective evaluation of the incidence of
inhibitors in patients treated with plasma-derived high
purity concentrate, first generation, and second recombinant
products suggested an increased incidence of inhibitors
using later products. This increased immunogenicity is
hypothesized to be secondary to alterations in posttransla-
tional modifications of FVIII and a reduction of von Will-
ebrand factor binding.48–51 These findings were challenged
by two systematic reviews52,53 and a patient-level meta-
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analysis.46 Then, a series of ad hoc analyses of national
untreated HA cohorts54–56 showed a significant difference
of inhibitor incidence among recombinant products. This
evidence was recently supported by findings from a national
cohort.57 The only randomized trial so far available strongly
supported the concept of a higher inhibitor incidence among
untreated patients using recombinant products.58 On the
other hand, the current evidence does not show this
increased risk in patients after the first 50 to 75 exposure
days, suggesting that factors leading to inhibitors develop-
ment are likely to play their role only early during the first
exposure days.
Final Considerations
Overall, available data are fairly consistent and suggest that a
series of potentially modifiable risk factors exist that can be
modulated with the aim of lowering the occurrence of
inhibitors.
The Multifactorial Model
Since its introduction, the multifactorial threshold model has
been widely applied in genetic counselling for qualitative
(discrete) traits.59–61 The multifactorial model includes the
influence of many factors, genetic and environmental. The
risk does not change drastically with the presence or the
absence of one of them. This implies that the susceptibility to
inhibitor formation is largely accounted for by the clustering
of several, possibly inherited, risk factors. Usually, geneticists
admit that in a multifactorial phenotype, several loci, but not
an unlimited number, are involved in the expression of the
trait; there is no classical (Mendelian) dominance or reces-
siveness at each of these loci; the loci act in concert in an
additive fashion, each adding or detracting a small amount
from the phenotype, and the environment interacts with the
genotype to produce the final phenotype. None of these
factors is either necessary or sufficient for the occurrence
of inhibitors, but the totality makes inhibitor development
more likely. This means that the liability to develop an
inhibitor-prone phenotype is continuously distributed in
the HA population because of the additive effects of genetic
 Inhibitor Development as a Multifactorial Event
and environmental factors. Our current thinking is that only
in individuals whose liability exceeds a certain threshold will
inhibitors then occur. However, because the phenotype alone
may not be able to distinguish different subgroups, genetic
heterogeneity may affect the ability to identify genetic risk
factors associated with inhibitor formation.
On the basis of the above-mentioned model, the
advancement in the understanding of risk factors that
underlie the occurrence of inhibitors fostered the search
for the creation of a clinical scoring system to quantify the
risk of each patient. In HA, a clinical scoring system using
three factors (F8 gene mutation, family history of inhibitor,
and intensive treatment at initial exposure) to predict
inhibitor formation identified that patients without any
risk factors (risk score ¼ 0 points) had an inhibitor inci-
dence of 6%, whereas those with two or more than three
points showed an incidence of 23 and 57%, respectively.62 A
different clinical scoring system showed rates ranging from
4% in the most favorable combination (O blood type, F8 low
risk mutation, and use of plasma-derived FVIII products) to
nearly 70% (non-O blood type, high-risk mutation, and use
of recombinant FVIII products).36 Finally, a 15-item predic-
tion score model using a 5-point linear scale (with “1” being
very unlikely and “5” very likely) found important factors
for inhibitor development, including genetic factors (mean
score: 4.50) and early intensive treatment (mean score:
4.07), as well as important management strategies to
reduce inhibitor development, including early onset of
prophylaxis (mean score: 3.54) and avoidance of elective
surgery (mean score: 4.32).63 The retrospective nature of
the analysis hardly hampers the use of these clinical scoring
systems. In addition, none of the clinical scoring systems
Fig. 1 Schematic representation of most represented “omics” technologies.
Margaglione, Intrieri
allows for a timely estimation of the individual risk when an
incidental risk factor happens.
Looking Forward
In recent years, new high-throughput technologies for assay-
ing biological activity using holistic approaches for biomo-
lecules such as DNA, RNA, and proteins have been developed,
where no hypothesis is known or proposed, but all data are
acquired and analyzed to generate hypotheses. Such large-
scale approaches have enabled unprecedented views of
biological systems at the molecular level (►Fig. 1). The fields
of research associated with obtaining and understanding
such measurements—for instance, genomics, transcrip-
tomics, and proteomics—are sometimes referred to in aggre-
gate as “omics.” As a whole, “omics” technologies allow for a
much more powerful approach to unravel key regulators
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leading to multifactorial conditions such as inhibitors
development.
Genome-wide expression profiling studies investigate the
activity of the genome rather than the inherent genome
variation and may identify which genes are “switched on”
and how much in any given situation such as when inhibitors
occur. In patients with HA, the CD4þ T cells–mediated
immunologic response is strongly related to the synthesis
of anti-FVIII antibodies.64 In the immunological response,
activation of CD4þ T cells regulates the proliferation and
differentiation of B cells and results in the acquisition of a
well-organized expression of multiple effector genes. Tran-
scriptional profiling could be investigated to assess
responses of CD4þ T cells, especially type-2 helper T cells,
to different exogenous formulations of FVIII and identify
Seminars in Thrombosis & Hemostasis
Inhibitor Development as a Multifactorial Event Margaglione, Intrieri
cell-specific responses which could predict inhibitor
development.65
It is well known that gene expression is modulated by
mechanisms other than the DNA sequence. Epigenetics
refers to dynamic changes in chromatin structure that play
a key role in regulating genome functions, including tran-
scription. On the whole, mechanisms such as DNA methyla-
tion and posttranslational modifications of histone proteins
play a significant role in the regulation of inducible immune-
responsive gene transcription. In CD4þ T cells, genes with
different basal expression levels are associated with specific
combinations of histone modifications and reorganization at
gene promoters and enhancers in response to T cell activa-
tion.66 Thus, cells depending on the phase of their life possess
distinct and characteristic patterns, “signatures,” of gene
expression and chromatin structural adaptation.
In general, genes with a low expression rate are related to
promoters with repressed chromatin signatures, whereas an
active chromatin signature shows intermediate to high gene
expression levels.67 In HA, understanding how the exposure
to FVIII modulates the dynamic interplay between hetero-
chromatin and euchromatin formation leading to chromatin
reorganization and, in turn, to the regulation of gene expres-
sion is expected to be crucial for the identification of key
mechanisms for the occurrence of inhibitors. Thus, a combi-
nation of transcriptional and epigenetic profiling to study
both gene expression and chromatin signatures, together
with the additional help of in silico information from bioin-
formatics, computational modelling, and pathway analysis,
will boost our knowledge of how inhibitors develop and in
response to which stimuli.
Final Considerations
Cutting edge technologies may enable a thorough and clever
estimation of the personal risk profile, which is currently
relatively inaccurate, involving the assessment of “omics”
signatures or biomarkers. This approach will represent a
welcome improvement in the ability to measure the risk for
inhibitors development in a meaningful way.
Conclusion
There is good evidence that host genetic factors are impor-
tant, but not the only determinants of the immunologic
response to FVIII administration. Genetic studies demon-
strated that many responses are partially predictable, and
modifiable factors modulate the appearance of inhibitors.
The comprehension of mechanisms leading to inhibitors
formation will pave the way for the complete understanding
of how risk factors determine their development. It can be
anticipated that different risk factors may interact each other
and give raise to an epistatic effect. Indeed, very recently it
was suggested that the use of recombinant products gives a
paradoxical high risk in patients carrying low-risk F8 gene
mutations. Thus, it is conceivable that the specific personal
array of nonmodifiable and possible modifiable risk factors
may identify a unique risk profile for each patient with
hemophilia. The personal risk profile may change over
Seminars in Thrombosis & Hemostasis
time, depending on the appearance of transient risk factors.
If it will become possible to have a reliable tool to accurately
measure from time to time the risk, it will become realistic to
have strategies to foresee and prevent inhibitor formation.
Conflict of Interest
None.
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