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Perspectives ingenomics andsickle cell disease therapeutics 193
https://t.me/med1917
Management of SCD
Effective management of SCD involves early detection via
NBS, followed by comprehensive preventive care. There are
relatively few reported specialized SCD treatment centers
across SSA (Figure14.3A and B) that offer standard- of- care
treatment for SCD, involving family education on primary
preventative measures and home- based care, such as the recognition of when to seek medical care, how to detect an
enlarged spleen, and prevention of dehydration by high fluid
intake. HU remains the most extensively tested therapeutic
(with over 100 ongoing or completed trials listed in http://
ClinicalTrials.gov), and the best- known disease- modifying
therapy for SCD recommended for all SCD children from
nine months of age and adults. Clinical trials– notably the
REACH, and NOHARM trials – have demonstrated the
safety and efficacy of HU to treat SCD in Africa. A major
challenge with HUT has been the determination of optimal
dosing and monitoring regimens, especially given the high
interpatient variability in HU response. Previous clinical trials, for instance in Europe, using a fixed dosage of 15–20 mg
per kilogram per day (mg/kg/day) have proven the effectiveness of HU with only a few reversible toxicities including
mild to moderate neutropenia, followed by reticulocytopenia and then thrombocytopenia. The REACH and NOHARM
dose- escalation trials in select settings in Africa have now
shown that even higher levels of HbF (>30% increase) can be
achieved by gradually increasing the HU dosage every two
months until a maximum tolerated dose (MTD) of ~35 mg/
kg/day. Recently, researchers have developed a new
pharmacokinetic- guided precision dosing method in children that reduces the time to determine a child’s MTD and
improves the HbF to >40%. The potential benefit of this
method is that it will significantly reduce the logistical challenges of the “trial- and- error” dose- escalation dosing
method, as well as laboratory monitoring and the need for
provider expertise. However, the method is yet to be tested in
adult SCD patients, and in Africa in general. Other potential
challenges to HUT in SSA are HU hesitancy and adherence
to treatment out of clinical research trials. In addition, there
is an ongoing discussion on whether longincreases exposure to reproductive effects such as infertility.
However, information on this subject remains scant, thus
emphasizing the need for longitudinal cohort (follow- up)
studies to accompany the implementation of HUT in SSA.
Blood transfusion is commonly used globally to manage
acute complications of SCD such as splenic sequestration,
hemolysis, as well as malaria- induced hemolytic anemia.
However, major challenges that hinder blood transfusion for
SCD patients in Africa include inadequate blood supply,
high risk of transfusion- transmitted infections such as bacterial and viral infections, which are usually high in the general
population, as well as transfusion- dependent hemolysis due
term HU usage
to the soantibodies against transfused RBCs (alloantibodies) destroy
the host’s (auto- ) RBCs. In addition, chronic transfusion can
lead to iron overload. Therefore, improvement of Africa’s
transfusion system such as increasing blood supply by
encouraging blood donation, improving blood screening
tests, improving donor–recipient matching, as well as
increasing accessibility to chelating agents in the case of
chronic blood transfusions will greatly reduce SCD morbidity and mortality.
Hematopoietic (allogenic) HSCT is currently the only
curative measure for SCD, with >95% cure rate in HLAmatched sibling donors. When performed in time, HSCT
can completely restore marrow function in SCD patients.
Immunological reactions like graft- versus- host disease
(GVHD) limit the use of unrelated matched donors, but
could be alleviated by gene therapy or gene editing (autologous HSCT). In Africa, the existence of large families could
have favored HSCT by the availability of matched sibling
donors. However, access to, and costs of HSCT remain challenging in the continent. The process and techniques
required for performing these interventions such as harvesting of autologous HSCs (CD43+) require specialist training
that is scarce in Africa. Only two countries in SSA have
HSCT centers: Nigeria and South Africa. Tanzania has put
forward an agenda to establish a center (Figure 14.3C).
Increasing funding by suband international organizations is required to invest in HSCT
centers and further clinical trials in Africa. This is particularly important as genetic studies on the continent are
increasing shedding light on Africa- specific genetic factors
in SCD.
called innocent bystander mechanism whereby
Saharan African governments
Current known genetic determinants
ofoutcomes insickle cell disease
The interaction of the HbS allele with other red cell genetic
variants that also confer malaria resistance and that are also
prevalent in malariagenetic interactions that either increase or reduce SCD clinical severity (Figure 14.4A–D). For instance, the common
3.7 kb α- thalassemia deletion that is particularly prevalent
among patients with SCD in Africa (Figure14.4B) compared
to ethnolinguistically matched control populations, is
associated with better SCD hematological indices and low
hospitalization rates, while the G6PD deficiency variants
(Figure 14.4C) which protect against some severe malaria
forms in SSA are associated with increased risk of hemolytic
anemia in SCD. The APOL1 G1 and G2 variants, which are
prevalent in SSA due to the protection they afford against
African trypanosomiasis (sleeping sickness), are also known
to predispose individuals of African ancestry – including
SCD patients– to end- stage renal disease (Figure14.4D).
endemic regions has resulted in complex
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194 Molecular Hematology
> 0.136
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(A) (B)
HbS
0-0.024
0.024-0.047
0.047-0.071
0.071-0.094
0.094-0.118
0.118-0.141
0.141-0.163
0.163-0.181
> 0.181
(C) (D)
G6PD
0-0.018
0.018-0.035
0.035-0.053
0.053-0.071
0.071-0.088
0.088-0.106
0.106-0.122
0.122-0.136
α-thalassemia
0-0.036
0.036-0.073
0.073-0.109
0.109-0.146
0.146-0.182
0.182-0.218
0.218-0.252
0.252-0.279
> 0.279
APOL1-G2
0-0.019
0.019-0.037
0.037-0.056
0.056-0.075
0.075-0.093
0.093-0.112
0.112-0.129
0.129-0.144
> 0.144
Figure14.4 Co- evolution of the HbS allele and other gene variants in Africa. (A) Distribution of the HbS allele. (B) Distribution of the 3.7 kb
alpha- thalassemia variant. (C) Distribution of the G6PD deficiency. (D) Distribution of the APOL1 G2 variant.
The HBB gene cluster haplotypes (Senegal: SEN, Benin: BEN,
Cameroon: CAM, Central African Republic: CAR, and
Arab- India: AI) (Figure 14.2) which are present in all
βS- bearing chromosomes are also associated with variable
SCD clinical severity: the AI, SEN, and BEN haplotypes
being associated with less severe SCD clinical outcomes, while
the CAM and CAR haplotypes are associated with poorer
outcomes. A recent study based on sequence data, rather
本书版权归John Wiley & Sons Inc.所有
than restriction site data, however, found sub- structuring
among the haplotypes that may have confounded their association with SCD clinical severity in some populations implying that further research is needed to understand the exact
role of the different haplotypes in SCD clinical outcomes.
The strongest known modifier of SCD clinical outcome,
however, remains the level of fetal hemoglobin (HbF) – a
highly heritable trait– in adult blood. Three major loci have
Perspectives ingenomics andsickle cell disease therapeutics 195
(A)
εδ
https://t.me/med1917
Chr. 11 β-like genes
HS5 432
1 ψβ2 ψβ2
LCR
Embryonic Fetal Adult
Chr. 16 α-like genes
(B)
HS-40
γ
α
ζ2 ζ1
αγ
Increasing HbF production
Foetal reactivation gene editing/therapies
Small molecule targets for HbF modifying genes
miRNA treatment
mRNA therapy
BCL11A
Gγ Aγ
ψα ψα α2 α1 θ
α
Increasing HbA production
mRNA therapy
Sickel cell disease-
causing mutation
β
αβ
β
Figure14.5 Genomics approaches for therapy aiming at hemoglobin productions. (A) Hemoglobin gene clusters. During fetal life, fetal hemoglobin
(HbF; α2γ2) is the dominant form of hemoglobin, but after birth, the level of HbF decreases progressively to reach about 1% of its level prior to
birth and it is replaced by adult hemoglobin A (HbA; α2β2). The hemoglobin proteins (Hb) are tetramers with HbA being composed of two
beta(like)- chains produced by genes within a cluster on chromosome 11 and two alpha(like)- chains from genes on chromosome 16; and HbF
being composed of two gamma(like)- chains produced by genes on chromosome 11, as well as two alpha(like)- chains. Sickle- cell disease is caused
by a single nucleotide substitution in the beta- globin gene on chromosome 11. The regulation of Hb production is tightly controlled by repressive
transcription factors such as BCL11A and ZBTB7A, which particularly bind to the HbF gene promoters. Genes encoding chains for embryonic
hemoglobin are also highlighted, as well as pseudogenes in the globin gene clusters and enhancer sequences. (B) Potential genomics therapeutic
approaches for Sickle cell disease can involve reactivating HbF production with gene editing, small molecule, or miRNA treatment by inhibiting
repressive transcription factors. For therapeutics to increase production of either HbF or non- sickled HbA, it is possible to use RNA therapy in a
similar approach used for the effective and successful development of COVID- 19 vaccine.
been identified to influence HbF levels: XmnI- HBG2
(11p15.4), BCL11A (2p16.1) (Figure14.5), and HBS1L- MYB
(6q24). Polymorphisms in these loci are associated with variable levels of HbF (1–30%) in adults. Functionally relevant
variants have been proposed for each of the loci: the XmnI
restriction variant (rs7482144) at HBG2 (frequent in populations in the Middle East), the rs1427407 variant at BCL11A
(the most influential HbF modifying locus in Africa), and
the 3 bp deletion rs66650371 at HBS1L- MYB. BCL11A is a
transcriptional repressor of HbF, with a well- characterized
HbF- silencing role in adults, and is therefore the target of
HU, the most widely used SCD therapeutic. Other loci
recently associated with HbF levels include BCL2L1
(20q11.21), which is yet to be replicated in Africa,
and FRMPD4 (Xp22.2) among Tanzanian SCD patients.
Most studies on HbF- promoting loci have been based on
non- African populations, and explain up to 50% variation of
HbF levels, meaning that a lot remains to be known about
other potential loci in SSA populations. Recently, wholeexome sequencing (WES) has uncovered variants in multiple
genes including in CLCN6, SERPINC1, ATP2B4, and
OGDHL linked to prolonged survival without complications
requiring therapy in Cameroonian SCD patients. Variants in
other genes (COL11A1, ABCC1, INSR, as well as ATP2B4)
were associated with increased susceptibility to stroke.
Comprehensive care can be achieved by also considering the
genetic factors that predispose some individuals to variable
clinical outcomes. African populations carry the highest
amounts of genetic diversity, with often more extreme disease phenotypes, which could significantly increase the
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196 Molecular Hematology
https://t.me/med1917
sensitivity of genetic studies in identifying disease- associated
variants on the continent.
Exploring themissing heritability
offetal hemoglobin inAfrica
touncover therapeutic targets
During fetal life, fetal hemoglobin (HbF; α2γ2) is the dominant form of hemoglobin (Figure 14.5A). After birth, the
level of HbF decreases progressively to roughly 1% of its level
prior to birth, in 8–12 weeks, and is replaced by adult hemoglobin A (HbA; α2β2). The regulation of Hb production is
tightly controlled by repressive transcription factors, such as
BCL11A and ZBTB7A, that preferentially bind to the HbF
gene (HBG) promoters (Figure14.5A). Recent studies identified another HbF repressor gene, ZNF410, which activates
the expression of Chromodomain Helicase DNA Binding
Protein 4 (CHD4), a component of a repressor complex that
is recruited to the γ- globin promoter by BCL11A and
ZBTB7A. Genetic variations in HbF- modulating genes allow
some individuals the capacity to continue producing HbF in
adult life. Because of stressed erythropoiesis, to compensate
with the recurrent hemolysis and related anemia, the expression of HbF in patients with SCD is higher compared to the
general population. However, SCD patients that further
retain the capacity of producing much higher levels of HbF
(>8%) after birth have fewer disease complications and
longer life expectancy because the presence of HbF in sickle
RBCs delays deoxy- HbS polymerization, and thus reduces
clinical complications.
There have been important recent advances in gene editing curative therapies for SCD, with two distinct approaches:
First, gene addition, which involves introducing a new gene
into the patient to be integrated and expressed, for example,
targeting mutated beta- globin gene to boost the production
of non- sickled HbA, or adding a gene with antisickling properties. For instance, the first gene therapy treatment of SCD
was reported in 2017, and used a Lentiviral vector with T87Q
mutation (HbAT87Q), that encodes a modified βgene, HbAT87Q, leading to steric inhibition of HbS polymerization. After nearly 20 months of follow- up, the median
total hemoglobin level increased from 8.5 g/dL at baseline to
≥11 g/dL in 35 patients. Second, gene editing which aims to
modify the native gene itself. A highly successful geneediting strategy for treating individuals with a double copy of
the sickle mutation (HbSS disease, or HbS- beta- zerothalassemia), aims at targeting transcriptional repressor such
as BCL11A to reactivate HbF, with CRISPR Cas- 9 disruption,
CRISPR- Cas- 12mutation of the HGB 1 and 2 enhancer sites,
and RNAi- induced suppression of BCL11a mRNA transcription through a short hairpin RNA expressed through a
lentiviral- based vector. Very promising results of a phase
globin
I study involving six patients with SCD showed a robust and
stable HbF induction in all patients (percentage HbF/[F
ranged from 20.4% to 41.3%). Clinical manifestations of
SCD were reduced or absent during the 6- to 29- month follow- up period. Both gene addition or editing require that the
HSCs be collected and harvested from the SCD patient.
Genetic modification then ensues invitro, followed by transplantation back to the patient after the residual marrow population is ablated using chemotherapy, with the major
advantages of autologous HSCT over allogeneic HSCT. It is
too early to evaluate which of gene addition versus Gene
editing is best; while waiting for longer- term follow- up data,
there are definitively more potential options/targets for reactivating HbF.
Indeed, it is estimated that more than 80% of gene variants
accounting for heritability of enhanced HbF expression after
birth are unknown in African populations (Figure14.6A).
The variants in the currently known HbF- modulating genes/
loci, including BCL11A, intergenic region HBS1L- MYB and
the HBB locus, explain up to 16% of enhanced HbF expres-
sion after birth in African individuals with SCD. This is compared with nearly 50% of the variants leading to HbF
persistence in adult Europeans being known (Figure14.6B).
This could mean that more variants in other HbF- controlling
gene/loci are still to be discovered in African populations.
Furthermore, the study that discovered the most currently
known modulator of HbF, BCL11A, was performed in populations of European ancestry, using Genome Wide
Association (GWAS) DNA arrays designed for that population. However, these DNA arrays do not capture the high
genetic diversity of understudied African populations.
Indeed, there is evidence that variants identified in GWAS
studies using UK Biobank samples led to the development of
polygenic risk scores associated with quantitative traits such
as blood indices, which then performed very poorly in populations with African ancestry. Moreover, very few GWAS
have been performed in African populations, which make up
only 2.5% of participants in currently available GWAS, globally. However, the limited African participants in GWAS
account for nearly 8% of all phenotype/disease associations
in this study. This high yield of data in the few GWAS that
included African populations is due to the high genetic
diversity in African people, the oldest population of humanity that has accumulated over 300
evolutionary history. Indeed, millions of genetic variants are
either more common, rarer, or specific to African populations, which also makes fine mapping of variants to disease/
trait associations more productive. Therefore, expanding
genomic research in populations of African ancestry, with
appropriately designed GWAS arrays that capture the extent
of genetic variation in that population, could uncover the
missing heritability of HbF- promoting loci. Of note, the only
GWAS performed for HbF level in about 1000 African
000 years of human genome
+ S]
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Perspectives ingenomics andsickle cell disease therapeutics 197
(A)
BCL11A (10%)
B
Africans Europeans
https://t.me/med1917
(B)
BCL11A
HBS1L-MYB (5%)
HBB locus (1%)
Missing
heritability
Missing
heritability
84%
Figure14.6 Missing heritability of HbF. Differential proportion of HbF variance attributed to each locus among Africans (A), compared to Europeans (B),
suggests that new loci that could be targets of HbF- manipulation therapies are more likely to be found among Africans.
56%
(15%)
HBS1L-MY
(19%)
HBB locus
(10%)
individuals living with SCD from Tanzania did not uncover
any novel HbF- modifying loci. This could suggest that additional modifier genes are likely to be rare and/or have low
effect sizes. Alternatively, due to the wide extent of unidentified variations in African populations, the GWAS array was
not suited to the genomic architecture of that Tanzanian
population. To uncover novel HbF- modifying loci, future
studies should use purposefully designed GWAS Arrays
developed from diverse African Genomes Sequences such as
the one designed by the H3Africa Consortium. That Array is
already showing some promising results with the discovery
of new loci for another quantitative trait, i.e. a novel LDL- C
association in the GATB region, while replicating several
well- known lipid- trait loci including LDLR, PMFBP1, and
LPA . The transferability of signals detected in two large
global studies consistently improves with an increase in the
size of the African replication cohort. The huge genetic
diversity and related complex haplotype structures in African
populations are illustrated by the consistent discovery of millions of new variants, suggesting that to better our understanding of HbF heritability and other complex traits,
multicentric studies including 1000s individuals from major
ethnolinguistic groups and diverse geographical regions
from in Africa will be needed. Moreover, deep sequencing of
suggestive loci should be performed systematically, followed
or complemented by in vitro functional studies in cell and
animal models, and extended to the innovative domainfocused CRISPR screen technology which has allowed the
identification of ZNF410, as well as HRI, an erythroidspecific kinase that controls HbF translation in humans, as
an HbF repressor. Ultimately, investigating fetal hemoglobin
genomics at scale in African populations, with comprehensive
functional analysis, will provide novel druggable targets for
effective HbF induction, either via gene editing therapy or
small molecules targeting those genes (Figure14.5B).
Investigating the prospect of RNA
therapy for SCD
Most RNA therapies can be sorted into one of three broad
categories: those that target nucleic acids (either DNA or
RNA), those that target proteins, and those that encode proteins. In general, the use of RNA therapy has a few advantages: first, most RNAs (miRNAs or mRNAs) are naturally
occurring molecules in human cells, with available mechanisms for their processing in place as well as and downstream
target selection. Second and specifically, miRNAs potentially
act by targeting multiple genes within one pathway, thus
causing a broader yet specific response, and mRNAs- based
therapy act on in- situ protein production, with an outstand-
ing safety profile, and exceptional flexibility. Third, unlike
gene editing/therapy RNA does not interact with the recipient genome, with potential unwanted off- target integration
for example in oncogenes, such as vector- induced leukemia
through enhancer- mediated mutagenesis in 25% of patients
in clinical trials for a rare genetic condition affection the
functions of white blood cells called X- linked severe combined immunodeficiency.
When it comes to short- term therapeutic potential for SCD,
non- coding microRNAs (miRNAs), which are strands of
18–25nucleotides and function to disrupt the production of
proteins by binding to the transcription machinery in a cell,
are the most promising (Figure14.5B). While recognizing that
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198 Molecular Hematology
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much is still unknown about the mechanism of action of HU,
the only widely available medication for SCD, there is consistent in vitro and invivo evidence suggesting that HU induces
HbF via miRNAs. Among adult patients with SCD living in
Africa, we found that the majority of miRNAs that are differentially expressed in response to HU treatment were functionally associated with HbF- regulatory genes, including BCL11A
(miR- 148b- 3p, miR- 32- 5p, miR- 340- 5p, and miR- 29c- 3p).
The response to HU and subsequent miRNA expression were
correlated with increasing HbF levels at baseline doses (for
miR- 494) and maximum tolerated doses (for miR- 26b and
miR- 151- 3p) of HU. Moreover, the use of miRNAs will allow
the ability to target an entire pathway of HbF production, with
a stronger cumulative output compared with targeting a single
gene. Research identifying more candidate miRNAs that act
on HbF production will provide an attractive new route for
future SCD therapeutics that mimic HU- induced HbF production, while minimizing potential HU effects on the whole
cellular transcriptome that could result in potential side
effects, like abnormal spermatogenesis. A second option of
RNA therapy for SCD is to mediate direct production of HbF
or non- sickled HbA through injection of exogenous mRNA,
which functions similarly to some recently developed
COVID- 19 vaccines (Figure14.5B). One major challenge for
this mRNA treatment is the delivery to the target organs and
cells. However, this issue is less prominent in SCD due to the
highly accessible bone marrow tissue where RBCs are generated. Moreover, blood is a renewable organ– in a normal individual RBCs are renewed every 120 days, and every 20 days in
patients with SCD– which allows an exit strategy if the mRNA
therapy causes unpredictable and unwanted results. The delivery mode of this treatment could utilize lipid nanoparticles or
polysaccharide- based nanoparticles that not only provide a
shield, but also harness existing cellular transport mechanisms
to get the nanoparticle and its cargo into the bone marrow cell.
However, it will be appropriate to underline the potential
challenges associated with RNA therapy and its delivery
approaches. Therapeutic trial results targeting nonRNAs (ncRNAs), such as microRNAs (miRNAs), have so far
been ambivalent, with some studies reporting potent effects,
whereas others demonstrated limited efficacy or toxicity.
Regarding the delivery, it is known that most nanoparticles
preferentially target the liver cell (hepatocytes), and most
microRNAs have numerous target genes. Therefore, intravenous, or intraosseous injection of nanoparticles encoding
miRNAs that target BCL11A, MYB, or other gamma- globin
gene regulators are likely to have off- target effects affecting
such as the liver function, and possibly other organ tissues.
For example, studies in mice showed that miRNA therapy
can be toxic, showing dose- dependent liver injury, ultimately
causing death in numerous experiments. Therefore, for the
purpose of future therapy, it will be imperative to controlling
the intracellular RNA expression levels. Other challenges
coding
specific to SCD is that targeting MYB in hematopoietic stems
cells and progenitors is likely to impair the overall hematopoiesis, affecting other blood cell beyond the RBC production. Alternative entities such as antimiRNAs are undergoing
clinical testing, and lncRNA- based therapeutics are gaining
interest. Encouragingly, at least 11 RNA- based therapeutics
are approved by the FDA and/or the European Medicines
Agency (EMA), aiming at gene modifications in liver, muscle, or the central nervous system, and numerous RNA therapeutics are in phase II or III clinical development. Moreover,
packaging mRNA into an adenovirus vector has been shown
to be an efficient delivery approach. For example, in 2018, in
both the United States and Europe, two RNA- based therapies
were approved for hereditary Transthyretin amyloidosis,
which is a progressive and potentially fatal disorder in which
abnormal proteins build up in nerves and organs, such as the
heart. Much recently, authors successfully suppressed heterotopic ossification in fibrodysplasia ossificans progressive,
an ultra- rare genetic disorder, using a combination of AAV
gene delivery, and miRNA silencing in a mice model. In
addition, atypical RNA interference effectors delivered by
AAV are capable of reducing, invivo, the disease severity of
retinitis pigmentosa due to Rhodopsin gene mutations.
These examples of therapeutic prospects urge future investment of resources and effort into the development of RNA
treatment for millions of patients living with SCD,
worldwide.
Developing genetic risk models for
SCD complications
Implementation of NBS and comprehensive care has led to a
drastic drop in SCD childhood mortality in the United States.
However, mortality in adults has not changed over the past
four decades in the United States due to patients developing
acute and chronic cardiovascular complications, such as
stroke and kidney disease, with the risk of these complications also being affected by genetic variations, such as variants in the APOL1 gene, or deletions in alphaSpecifically, genetic coding variants in APOL1, which encodes
apolipoprotein L1 (APOL1), were evolutionary selected in
populations with African ancestry to confer resistance to
trypanosome and to prevent sleeping sickness. Unfortunately,
these variants, known as G1 and G2, are a frequent cause of
the kidney disease, APOL1- nephropathy, in both patients
with SCD, and in the general population with African
Ancestry. Approximately, 10–30% of African Americans
and West/central Africans carry two APOL1 risk alleles.
Interestingly, recent research has provided compelling data
for novel therapeutic targets that may be useful for treating
APOL1- nephropathies. Currently, antisense oligonucleotide
drugs that inhibit APOL1 synthesis or function are in preclinical
globin gene.
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Perspectives ingenomics andsickle cell disease therapeutics 199
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and clinical testing. Therefore, there is an urgent need to
identify the full spectrum of genetic variants that modify
clinical complications of SCD, including variants associated
with long- term survival in unfavorable environments of
Africa, such as the recently identified variants in genes of the
- glutamine production pathways, and variants evolutionarily selected and/or co- inherited with the SCD mutation. We
observed in the “long survivors” group, defined as patients
living in Africa over >40without consistent access to modern
treatment, a high mutational burden in CLCN6 and OGHDL.
It was previously reported that rare, deleterious mutations in
CLCN6 (a voltage- dependent chloride channel) have been
associated with lower blood pressure. Given that increased
blood pressure is a major risk factor for stroke in SCD, the
result suggests that SCD patients with CLCN6 some specific
variants live longer due to a reduced risk of stroke. Similarly,
OGHDL is important in arginine metabolism, which is a key
factor in the hemolysis- endothelial dysfunction observed in
SCD and has become a target for FDA- approved therapeutic
interventions for the treatment of SCD. Both results and other
similar have the potential for informing anticipatory guidance in clinical practice, while pointing to targets for the
design of new SCD therapeutics.
Gene–environment interactions must also be considered
when developing novel therapeutics for SCD because of
important gene variants evolutionary selected in Africa as a
result of endemic infectious disease such as variant in G6PD,
or α- thalassemia 3.7 kb deletion associated with malaria
resistance and protective for kidney dysfunctions in SCD, or
variants in APOL1 protective against trypanosome but
increasing susceptibility to kidney dysfunctions in SCD.
Because these variants, like the SCD mutation, are frequent
enough in Africa, there are highly likely to be co- inherited by
numerous patients living with SCD. Therefore, systematic
evaluation of their interactions to influence the overall clinical severity of SCD is needed. Moreover, there is a large
inflammatory component associated with SCD pathophysiology, emphasized in Africa, where serious infections are
most common, including high bacteremia rates in SCD
patients. Thus, investigating gene–environment interactions
is even more relevant for both genetic associations in all individuals with SCD in Africa.
Understanding genetic variation in African populations
has advanced greatly in the past few years, with multiple
cross-
ancestry studies of common diseases or health- related
traits being performed in non- SCD patients, but with relevance to SCD. For example, in large African ancestry cohorts,
the APOL1 risk genotype and polygenic component of the
genome- wide polygenic score had additive effects on the risk
of chronic kidney disease. Other study found in UKBB participants seven novel signals in African ancestry, including a
cis pQTL for the gene encoding gamma- glutamyl transferase
and PIEZO1 and G6PD variants with impacts on HbA1c
through likely erythrocytic mechanisms. All these studies
should be expanded and employed in SCD patient cohorts,
with multicenter longitudinal studies using methodological
approaches, including classical GWAS, whole exome, and
whole genome sequencing, alongside multi- omics such as
transcriptomics, metabolomics, proteomics, and metagenomics. Knowledge gained from these multicenter longitudinal multi- omics studies in Africa will allow development of
mathematical models for genetic risks, enabling SCD patient
stratification in infants to cluster by severity and to optimize
treatment and care, accordingly.
Conclusion andperspectives
Sickle cell disease preventive measures including primary prevention through NBS and genetic counseling focused on individuals with sickle- cell trait will greatly reduce the prevalence
of the disease, mortality, and morbidly in Africa and globally.
Increased research in pharmacogenetics is needed to optimize
the anticipated increase in the number of SCD patients following widely available treatment such as HU, and other long- term
treatments for chronic complications. As HU dose- escalation
algorithms promise to further reduce SCD morbidity and mortality, many more SCD clinics in semi- urban and rural settings
need to be established in order to reach all SCD- affected individuals who do not have access to existing specialized treatment centers Africa. Finally, research is highly needed in Africa
in order to determine the exact prevalence, mortality and morbidity of SCD, environmental and genetic factors affecting
clinical complications, and life expectancy, in order to design
future risk models, and to investigate novel routes for therapeutic options, with the ultimate aim of improving SCD clinical
outcomes in all parts of the world.
Why should we invest in expensive genetic studies while
most countries in Africa where SCD is most prevalent are
not even able to implement basic care, such as prophylactic
penicillin? And how successful and equitable could such
genomic research be, considering the technological challenges and high cost associated with the currently available
hematopoietic transplant estimated cost of HSCT per patient
ranges from $350
as high as $1–2million?
In fact, considering that the total lifetime cost of managing
a patient living with SCD by age 50 exceeds $8million, in
comparison, the upfront high cost of HSCT or gene therapy/
editing could be acceptable. Therefore, a global effort to
explore new routes for therapeutics for SCD patients in all
parts of the world is essential. This will include the development of an invivo gene therapy delivery system that would
bypass the need for an autologous transplant and make
worldwide application possibly equitable. Moreover, investing in discovery novel therapeutic target, offers more option
000 to $800 000, and gene editing therapy
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200 Molecular Hematology
https://t.me/med1917
for reactivation of HbF using pharmacologic approaches
such as small molecule regulators for HbF modifying genes.
I believe that such effort is possible, as exemplified by the
recent fast development and implementation of COVID- 19
vaccine. Learning from recent failures in global vaccine distribution during the COVID- 19 pandemic, this effort should
be accompanied by a mechanism, through international
agencies such as the World Health Organization, to address
the equity crisis by establishing centers of excellence for SCD
care, particularly in Africa. This must be supported by concerted strategies from numerous stakeholders, including
industry, national governments, SCD patient support groups,
professional societies, international agencies, and funding
bodies with expanding mechanisms, such as the Cure Sickle
Cell Disease Initiative launched in September 2018 by the
National Heart Lung and Blood Institute, NIH, USA.
Exploring genomics in SCD could further our understanding of specific cardiovascular complications, such as stroke
or kidney disease, that are both common in SCD and in the
general population. Moreover, investing in genomics for new
SCD treatment development will serve as a mold for developing treatments for other monogenic diseases.
Further reading
Ataga, K.I., Saraf, S.L., and Derebail, V.K. (2022). The nephropathy of
sickle cell trait and sickle cell disease. Nat. Rev. Nephrol. 18 (6):
361–377.
Esoh, K. and Wonkam, A. (2021). Evolutionary history of sickle-
mutation: implications for global genetic medicine. Hum. Mol.
Genet. 30 (R1): R119–R128.
Esoh, K., Wonkam-
ease in subcare. Lancet Haematol. 8 (10): e744–e755.
Frangoul, H., Altshuler, D., Cappellini, M., D. etal. (2021). CRISPR-
Cas9 gene editing for sickle cell disease and β- thalassemia. N. Engl.
J. Med. 384 (3): 252–260.
Kanter, J., Walters, M.C., Krishnamurti, L. et al. (2022). Biologic and
clinical efficacy of LentiGlobin for sickle cell disease. N. Engl. J. Med.
386 (7): 617–628.
Nnodu, O.E., Oron, A.P., Sopekan, A. etal. (2021). Child mortality from
sickle cell disease in Nigeria: a modelanalysis of data from the 2018 demographic and health survey.
Lancet Haematol. 8 (10): e723–e731.
Wonkam, A. (2023). The future of sickle cell disease therapeutics rests
in genomics. Dis. Model Mech. 16 (2). dmm049765.
Tingang, E., and Wonkam, A. (2021). Sickle cell dis-
Saharan Africa: transferable strategies for prevention and
estimated, population- level
cell
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Chapter15
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Molecular coagulation
andthrombophilia
Björn Dahlbäck1 and Andreas Hillarp
1
Department of Translational Medicine, Lund University, University Hospital, Malmö, Sweden
2
Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway
Introduction, 201
Blood coagulation, 201
Regulation of blood coagulation, 202
Molecular genetics of venous thromboembolism, 206
2
Severe thrombophilia is a multigenic disease, 212
Management of thrombophilia, 212
Conclusions, 213
Further reading, 213
Introduction
two vitamin K-
Venous thrombosis is a major medical problem affecting
millions of individuals worldwide each year. It is a typical
multifactorial disease, the pathogenesis involving both environmental and genetic risk factors. A mutation in the factor
V (FV) gene (Arg506Gln or FV Leiden) is the most common
genetic risk factor known to date. Activated protein C (APC)
regulates the activity of FVa by cleaving several sites in FVa,
and Arg506 is one of them. APC resistance, which is the consequence of the FV mutation, results in a lifelong hypercoagulable state. A point mutation in the prothrombin gene is
another relatively common risk factor, whereas deficiencies
of the anticoagulant proteins antithrombin, protein C, or
protein S are less common. Owing to the high prevalence of
FV and prothrombin mutations, combinations of genetic
defects are relatively common in the general population.
Such individuals have a highly increased risk of thrombosis.
Blood coagulation
At sites of vascular damage, circulating platelets adhere to
subendothelial structures and undergo a series of reactions
that lead to primary hemostasis due to the formation of a
platelet plug. Concomitant to these events, the subendothelial membrane protein tissue factor (TF) is exposed to blood.
A small amount of activated factor VII (FVIIa) present in
circulating blood binds to TF and triggers a series of proteolytic reactions that culminate in the formation of thrombin
and the conversion of fibrinogen to insoluble fibrin.
and factor X (FX) (Plate15.1). Activated FX (FXa) activates
prothrombin to thrombin, whereas activated FIX (FIXa)
activates FX. Both FIXa and FXa are poor enzymes that
require protein cofactors, calcium ions, and negatively
charged phospholipid surfaces for the expression of full biological activity. The protein cofactors for FIXa and FXa are
the activated forms of factor VIII (FVIIIa) and factor V
(FVa), respectively (Plates15.1 and15.2). As a result of multiple protein–protein and protein–phospholipid interactions,
enzymatically highly efficient complexes are assembled on
the phospholipid surface.
referred to as the extrinsic pathway or the TF pathway. In
association with injury, this is the physiologically most
important mechanism of blood coagulation. However, coagulation can also be activated through the intrinsic pathway,
triggered by activation of the contact phase proteins (FXII,
FXI, prekallikrein, and high- molecular- weight kininogen)
that follows exposure of blood to certain negatively charged
surfaces. It has been discovered that platelets contain longchain negatively charged polyphosphates that may serve as a
natural activator for the intrinsic system. The intrinsic pathway does not appear to be physiologically important for
injury- related coagulation in vivo, illustrated by the lack of
bleeding problems in individuals with a deficiency of FXII.
However, the interest in the intrinsic pathway increased dramatically after the discovery that it may be important for the
formation of arterial thrombosis and efforts are made to
develop inhibitors to the system for safe anti- coagulation.
FVIIa bound to TF specifically cleaves and activates the
dependent plasma proteins, factor IX (FIX)
The initiation of blood coagulation by TF is usually
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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201
202 Molecular Hematology
(A)
A2
A2
Membrane
Å
(B)
Activation and propagation of coagulation
Extravascular cell Activated platelet
https://t.me/med1917
VWF
V
IXa
VIII
VIIIa
IXa
T
T
X
Xa
Va
Xa
PT
T
VIIa
TF
Plate 15.1 The initiation and propagation of blood coagulation. Blood coagulation takes place on the surface of cell membranes, where
enzymes and cofactors form complexes that efficiently convert their respective proenzyme substrates to active enzymes. The exposure of tissue
factor (TF) to blood with subsequent binding of FVII/FVIIa and activation of FIX and FX initiates the reaction sequence. The subsequent assembly of
tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes on the surface of negatively charged phospholipid membranes (mainly provided by
platelets) result in amplification, propagation of the process and in the generation of high concentrations of thrombin (T). Thrombin feedback
activates FVIII (circulates in complex with von Willebrand factor– VWF) and FV.
IX/X
IXa
Xa
IXa
Xa
R306 R306
A1
C2
A3
C1
Plate 15.2 Molecular model of FVa highlighting its interactions with FXa and APC. FVa is shown as a solid surface (domains A, green; A2,
cyan; A3, brown; C1, pink and C2 purple) and FXa as ribbon (in orange). The virtual membrane is represented as a grey box. (A) FVa- FXa complex
with FVa residues probed experimentally to be important for FXa binding being shown in white. (B) APC approaching the cleavage site at Arg506.
The two main cleavage sites of APC (Arg306 and Arg506) are indicated in red. The serine protease domain of APC as ribbon (dark blue). The
figure is from Segers K, Dahlback B, Nicolaes GA. (2007) Thromb Haemost. 98, 530–542; reproduced with permission. Recently, the 3D structures
of FV/FVa and the assembled prothrombinase complex determined by Cryo- em were reported (see Di Cera E etal, Res. Pract. Thromb. Haemost.
(2022)Nov 2;6(7):e12830. doi: 10.1002/rth2.12830).
R506
FXa
~70 Å
C2
A1
C1
Membrane
A3
(SP domain)
R506
~85
APC
Thrombin generated at sites of vascular injury expresses a
Regulation ofblood coagulation
number of procoagulant properties. It amplifies the coagulation process by activating FXI and in addition it activates
platelets and converts fibrinogen to fibrin. Moreover, in a
positive feedback reaction, thrombin converts the procofactors FV and FVIII into their biologically active counterparts
(FVa and FVIIIa).
The efficient reactions of the coagulation system have
considerable biological potential and strict regulation is
required. For this purpose, several plasma proteins and protein–cell interactions are involved in constant monitoring of
the circulation. At each level of the coagulation pathway,
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