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Beginnings: themolecular pathology ofhemoglobin 3
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The synthesis of hemoglobin tetramers consisting of two
unlike pairs of globin chains is absolutely essential for the
effective function of hemoglobin as an oxygen carrier. The
classical sigmoid shape of the oxygen dissociation curve,
which reflects the allosteric properties of the hemoglobin
molecule, ensures that, at high oxygen tensions in the lungs,
oxygen is readily taken up and later released effectively at the
lower tensions encountered in the tissues (Figure1.1D). The
shape of the curve is quite different to that of myoglobin, a
molecule that consists of a single globin chain with heme
attached to it, which, like abnormal hemoglobins that consist
of homotetramers of like chains, has a hyperbolic oxygen dissociation curve.
The transition from a hyperbolic to a sigmoid oxygen dissociation curve, which is absolutely critical for normal oxygen delivery, reflects cooperativity between the four heme
molecules and their globin subunits. When one of them takes
on oxygen, the affinity of the remaining three increases markedly; this happens because hemoglobin can exist in two configurations, deoxy(T) and oxy(R), where T and R represent
the tight and relaxed states, respectively. The T configuration
has a lower affinity than the R for ligands such as oxygen. At
some point during the addition of oxygen to the heme molecules, the transition from the T to the R configuration occurs
and the oxygen affinity of the partially liganded molecule
increases dramatically. These allosteric changes result from
interactions between the iron of the heme groups and various
bonds within the hemoglobin tetramer, which lead to subtle
spatial changes as oxygen is taken on or given up.
The precise tetrameric structures of the different human
hemoglobins, which reflect the primary amino acid
sequences of their individual globin chains, are also vital
for the various adaptive changes that are required to ensure
adequate tissue oxygenation. The position of the oxygen
dissociation curve can be modified in several ways. For
example, oxygen affinity decreases with increasing CO
tension (the Bohr effect). This facilitates oxygen loading
tothe tissues, where a drop in pH due to CO2 influx lowers oxygen affinity; the opposite effect occurs in the
lungs. Oxygen affinity is also modified by the level of
2,3- diphosphoglycerate (2,3- DPG) in the red cell.
Increasing concentrations shift the oxygen dissociation
curve to the right (i.e. they reduce oxygen affinity), while
diminishing concentrations have the opposite effect. 2,3DPG fits into the gap between the two β chains when it
widens during deoxygenation, and interacts with several
specific binding sites in the central cavity of the molecule.
In the oxygenated configuration, the gap between the
twoβ chains narrows and the molecule cannot be accommodated. With increasing concentrations of 2,3- DPG,
which are found in various hypoxic and anemic states,
more hemoglobin molecules tend to be held in the deoxy
configuration and the oxygen dissociation curve is
thereforeshifted to the right, with a more effective release
of oxygen.
Fetal red cells have greater oxygen affinity than adult red
cells, although, interestingly, purified fetal hemoglobin has
an oxygen dissociation curve similar to that of adult hemoglobin. These differences, which are adapted to the oxygen
requirements of fetal life, reflect the relative inability of Hb F
to interact with 2,3- DPG compared with Hb A. This is
because the γ chains of Hb F lack specific binding sites for
2,3- DPG.
In short, oxygen transport can be modified by a variety of
adaptive features in the red cell that include interactions
between the different heme molecules, the effects of CO2,
and differential affinities for 2,3- DPG. These changes,
together with more general mechanisms involving the cardiorespiratory system, provide the main basis for physiological
adaptation to anemia (Figure1.1).
The structure ofthe globin gene loci
The α- and β- like globin chains are the products of two different gene families which are found on different chromosomes (Figure1.1C). The β- like globin genes form a linked
cluster on chromosome 11, extending over approximately
60 kb (kilobase or 1000nucleotide bases). The different genes
that form this cluster are arranged in the order 5′–ε–Gγ–Aγ–
ψβ–δ–β–3′. The α- like genes also form a linked cluster, in
this case on chromosome 16, in the order 5′–ζ–ψζ–ψα1–
α2–α1–3′. The ψβ, ψζ, and ψα genes are pseudogenes; that is
they have strong sequence homology with the β, ζ, and α
genes, but contain a number of differences that prevent them
from directing the synthesis of any protein products. They
may reflect remnants of genes that were functional at an earlier stage of human evolution.
The structure of individual human globin genes is similar
to that of all mammalian genes. They consist of long strings
2
of nucleotides that are divided into coding regions, or exons,
and non- coding inserts called intervening sequences (IVSs)
or introns. The α- like globin genes contain two introns,
one of 117 base pairs between codons 31 and 32 (IVS1)
andone of 142–147 base pairs between codons 99 and 100
(IVS2). The exon codons are numbered sequentially from
the 5′ to the 3′ end of the gene, that is from left to right
(Figure1.1C). Similar introns are found in the other globin
genes, though notably IVS2 is much larger in the β- globin
gene, 850–904 base pairs. These introns and exons, together
with short non- coding sequences at the 5′ and 3′ ends of the
genes, represent the major functional regions of each gene.
However, there are also extremely important regulatory
sequences which control the expression of each gene. At the
5′ non- coding (flanking) regions of the globin genes, there
are elements that define the promoters of the globin
genes.The first, the TATA box, is about 30 bases upstream
4 Molecular Hematology
Excision of introns
(B)
(A)
chain
https://t.me/med1917
(to the left) of the initiation codon; that is the start signal for
the beginning of transcription. A second sequence, the
CCAAT box, is about 70 base pairs upstream from the 5′ end
of the genes. About 80–100 bases further upstream, there is
the sequence GGGGTG, or CACCC box. These three highly
conserved DNA sequences, called promoter elements, are
involved in the initiation of transcription of the individual
genes. Finally, in the 3′ non- coding region of all the globin
genes, there is the sequence AATAAA, which is the signal for
cleavage and polyA addition to RNA transcripts.
The globin gene loci each contain clusters of several short
(~200 bp) sequences lying 40–50 kb upstream of the globin
genes that constitute their major regulatory elements, which
interact with their cognate promoters to enhance erythroidspecific gene expression and coordination of changes in globin gene activity during development (Figure1.1C). In both
the α-
and β- clusters, there are several such elements collectively referred to as locus control regions (e.g. LCRs at the
β- globin locus) or super- enhancers (SEs at the α- globin
locus). The elements within each cluster have a modular
structure made up of arrays of short motifs that represent the
binding sites for transcriptional activators or repressors.
Enhancers thereby sense and coordinate the erythroid transcriptional and epigenetic programs and communicate this
information to the promoters. The mechanism(s) by which
they transfer information to the promoters is unknown but
under intense investigation.
Transcription, processing, and translation
ofglobin RNA
The activation of transcription from the globin genes
depends on signals initiated from the enhancers which bind
the key erythroid factors (GATA1, SCL, LMO2, E2F, LDB1,
KLF1, and NFE2) and physically contact the globin gene
promoters to activate their expression as erythropoiesis proceeds. The precise mechanism by which they do this is not
clear, but it may involve the Mediator complex and certainly
involves recruitment of the pre- initiation complex (PIC)
and RNA polymerase (PolII) to the globin gene promoters
(Figure1.2A).
The flow of information between DNA and protein is also
summarized in Figure1.2B. Globin gene transcription begins
with the formation of a transcription complex (the PIC),
which consists of a variety of regulatory proteins together
with the enzyme RNA polymerase II.
The primary RNA transcript is a large precursor which
contains both intron and exon sequences. While in the
nucleus, this molecule undergoes a variety of cotranscriptional processing changes. First, the introns are
removed and the exons are spliced together. The majority
of intron/exon junctions for all mammalian genes have a
consensus sequence: GT at their 5′ end and AG at their 3′
end. This appears to be essential for accurate splicing; if
there is a mutation at these sites, splicing does not occur
correctly. Splicing reflects a complex series of intermediary
Promoter
Chromatin bre
RNA Polymerase II
Promoter
Enhancer
Activators
Gene
Transcription
factors
C
A
C
C
C
Nucleus
Cytoplasm
Transfer RNA
C
T
C
A
A
A
T
T
A
G
A
T
5' 3'
5' CAP
U
Ribosome
A
U
U
A
Amino
acid
Processed
G
C
G
C
U
A
C
G
A
Growing
chain
C
UAA
U
G
A
Finished
chain
AAAA-A
AAAA-A
AAAA-A
A
A
T
T
A
A
A
A
A
FlankingFlanking IVS 1 IVS 2
NCAGGTAGGTNC
Gene
mRNA precursor
Splicing of exons
Processed mRNA
Translation
Figure1.2 (A) Looping at gene loci bringing enhancers, promoters, transcription factors, activators, the Pre- Initiation complex and RNA
Polymerase (PolII) into physical proximity. (B) The mechanisms of globin gene transcription and translation.
Beginnings: themolecular pathology ofhemoglobin 5
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stages and the interaction of a number of different nuclear
proteins (the spliceosome). After the exons are joined, the
mRNAs are modified and stabilized; at their 5′ end, a “CAP
structure” is formed, while at their 3′ end, a string of adenylic acid residues (polyA) is added. The mRNA processed
in this way moves into the cytoplasm, where it acts as a
template for globin chain production. Because of the rules
of base pairing— that is cytosine always pairs with thymine,
and guanine with adenine— the structure of the mRNA
reflects a faithful copy of the DNA codons from which it is
synthesized; the only difference is that, in RNA, uracil (U)
replaces thymine (T).
In the cytoplasm, amino acids are transported to the
mRNA template on carriers called transfer RNAs (tRNAs);
there are specific tRNAs for each amino acid. Furthermore,
because the genetic code is redundant (i.e. more than one
codon can encode a particular amino acid), for some of the
amino acids, there are several different individual tRNAs.
Their order in the globin chain is determined by the order
of codons in the mRNA. The tRNAs contain three bases,
which together constitute an anticodon; these anticodons
are complementary to mRNA codons for particular amino
acids. They carry amino acids to the template, where they
find the appropriate positioning by codon–anticodon base
pairing. When the first tRNA is in position, an initiation
complex is formed between several protein initiation factors together with the two subunits that constitute the ribosomes. A second tRNA moves in alongside and the two
amino acids that they are carrying form a peptide bond
between them; the globin chain is now two amino acid residues long. This process is continued along the mRNA from
left to right, and the growing peptide chain is transferred
from one incoming tRNA to the next; that is the mRNA is
translated from 5′ to 3′. During this time, the tRNAs are
held in appropriate steric configuration with the mRNA by
the two ribosomal subunits. There are specific initiation
(AUG) and termination (UAA, UAG, and UGA) codons.
When the ribosomes reach the termination codon, translation ceases, the completed globin chains are released, and
the ribosomal subunits are recycled. Individual globin
chains combine with heme, which has been synthesized
through a separate pathway, and then interact with one like
chain and two unlike chains to form a complete hemoglobin tetramer.
The regulation ofglobin gene transcription
Although there are many levels at which globin gene expression could be modified (transcription, RNA processing,
nuclear export, translation, and protein assembly), the levels
of globin expression in normal hematopoiesis are primarily
controlled at the level of transcription. In stem cells and
early progenitors, very little globin RNA is produced.
However, early in erythroid differentiation chromatin
containing the regulatory elements opens and becomes
accessible as pioneer factors such as GATA2 start to prime
the regulatory elements. These regulatory elements then
become increasingly open and bind the factors that specify
erythroid gene expression (GATA1, SCL, LMO2, E2F, LDB1,
KLF1, and NFE2). As cells become committed to erythroid
differentiation, the enhancers come into close proximity to
the promoters and facilitate the binding of the PIC and
Polymerase II. Transcription then begins and the chromatin
in the globin loci becomes acetylated indicating activation
(Figure1.2A).
Some degree of regulation is mediated by differences in
the stability of α- and β- mRNA, the rates of initiation and
translation of the different mRNAs, and at the posttranscriptional level by differential affinity for different protein
subunits. However, this posttranscriptional fineprobably plays a relatively small role in determining the
overall output of the globin gene products.
tuning
The regulation ofdevelopmental globin
switching
Erythropoiesis occurs in three independent phases during
development. The first wave of hematopoiesis (primitive
erythropoiesis) originates in the embryonic blood islands. In
these cells, only embryonic hemoglobins (Gower I ζ2γ2,
Gower II α2γ2, Portland ζ2γ2) and some fetal Hb (α2γ2) are
made (Figure1.1C). A second wave of hematopoiesis (called
EMP hematopoiesis) is derived from endothelial cells in the
embryo, and this represents the first wave of definitive erythropoiesis, which takes place in cells that migrate to the fetal
liver and in which the embryonic genes are now silenced.
EMP cells produce fetal Hb (α2γ2). The final wave of definitive hematopoiesis, which replaces EMPs, arises from
hemato- endothelial cells in the ventral wall of the dorsal
aorta to form multi- potent hematopoietic stem cells (HSCs),
which subsequently form all mature blood cells throughout
life; initially in the fetal liver and finally in the bone marrow.
These cells make fetal Hb (α2γ2) during fetal life and adult Hb
after birth.
Of great interest, globin gene expression is reflected in the
linear order of the genes along each globin cluster: embryonic, fetal and adult (Figure 1.1C). While it is still largely
unknown how the embryonic genes (ζ and ε) are silenced in
definitive erythropoiesis, a great deal has been discovered
about the fetal (γ) to adult (β) switch that takes place in the
permanent, definitive HSCs.
The embryonic genes are located in the middle of a highly
active globin locus, close to the fully functional enhancers
that normally activate the ζ and ε genes in embryonic life,
and yet these genes are almost completely silenced in normal
adult erythropoiesis. This suggests that there is a molecular
6 Molecular Hematology
Chr
BCL11A
Chr
Algerian HPFH
~13kb
~24kb
(B)
(A)
https://t.me/med1917
mechanism which actively silences these genes. Although
some of the factors that silence the γ- globin genes in adult
life (see below) are involved in silencing the embryonic
genes, this is not the full story and the mechanism involved
in silencing these genes is currently under investigation.
By contrast, regulation of the switch from γ- to β- globin
expression during normal development of HSCs is now well
understood. The key cis- acting elements in the γ genes were
largely identified via the analysis of families with mutations
causing non- deletional hereditary persistence of fetal hemoglobin (HPFH, see below). These elements lie within and
closely upstream of the γ- globin gene promoters. The mechanistic importance of these elements was not appreciated
until genetic association studies identified two transcription
Erythroid specific
enhancer elements
. 2
Exon 1
Exon 2 Exon 3 Exon 4 Exon 5
+55 +58 +62
factors BCL11A and ZBTB7a (also known as Pokemon and
leukemia regulatory factor LRF). It is now known that
BCL11A, which is expressed in many blood cell types, has an
erythroid- specific enhancer. BCL11A and LRF are known to
bind to specific elements within the core promoter of the γglobin genes (Figure1.3A). There they displace an activating
transcription factor (NF- Y) that binds the γ- globin CCAAT
box, prevent GATA1 binding, and likely exert further repressive effects through the action of the repressive Nucleosome
Remodeling and Deacetylase (NuRD) complex. A number of
other modulators of γ- globin expression have been discovered, such as HRI, LIN28B, HIC2a, NFIA and NFIX, HIC,
HIF1α and ZNF420, the majority of which act by affecting
the BCL11A pathway.
BCL11A
. 11
β-LCR
β-LCR
12345
BCL11A
12345
HPFH-1 (Black)
HPFH-2 (Ghanaian)
HPFH-3 (Indian)
HPFH-4 (Italian)
HPFH-5 (Sicilian)
South-East Asian HPFH
Kenyan HPFH
French HPFH
NuRD
complex
GATA1
βδγ γε
βδγ γε ψβ
~85kb
~84kb
~50kb
~40kb
~30kb
~22kb
~20kb
Figure1.3 (A) Simplified mechanism by which γ- globin is repressed during the fetal- to- adult switch. (B) Deletions within the β- globin locus
causing deletional HPFH.
Beginnings: themolecular pathology ofhemoglobin 7
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Molecular pathology ofthe globin
genes
The disorders of hemoglobin are overwhelmingly caused
by mutations within the respective α and β gene loci. They
produce a set of monogenic diseases with variable severity,
which can be split into two overlapping categories. The
first are the thalassemias, which result in reduced production of α- or β- globin chains. The second is the structural
hemoglobinopathies (such as the sickle cell mutation),
which result from alterations in the protein structure.
Some variants such hemoglobin E, fall into both categories
as the underlying mutation affects both protein structure
and RNA splicing, causing reduced levels of stable RNA.
Of note, there is also a category of variants that result in
elevated Hb F production, known as hereditary persistence
of fetal hemoglobin (HPFH) Figure 1.3. While generally
not pathological, these conditions do have significant
interactions with hemoglobinopathies, and to date, their
analysis has provided the basis for some of the most effective treatments available for thalassemia and sickle cell
disease.
Because many of these diseases have been selected for in
regions where malaria is or has been endemic, it is not
uncommon for an individual to inherit a gene for one or
other form of thalassemia and a structural hemoglobin
variant, for example the sickle variant and β thalassemia.
The heterogeneous group of conditions that results from
these different mutations and their interactions is summarized in Table1.1. Reference to a comprehensive database
of thalassemia mutations is provided in the further reading
section.
Table1.1 The thalassemias andrelated disorders
α Thalassemia γ Thalassemia
0
α
+
α
Deletion (−α)
Non- deletion (αT) εγδβ Thalassemia
β Thalassemia Hereditary persistence of fetal hemoglobin
0
β
+
β
Normal Hb A
“Silent” Non- deletion
Dominant Linked to β- globin genes
δβ Thalassemia
+ Aγβ+
(δβ)
0
(δβ)
0
(Aγδβ)
2
δ Thalassemia
Deletion
(δβ)°
Gγβ+
Unlinked to β- globin genes
The α thalassemias
Normal individuals have two linked α- globin genes on each
allele of chromosome 16 and their genotype can be written as
αα/αα. There are two major categories of α thalassemia, α+
and α0 thalassemia. In α+ thalassemia one of the linked αglobin genes is lost, either by deletion (−) or single- point
mutations (T); the genotype of carriers can be written −α/αα
or αTα/αα. In α0 thalassemia, the loss of both α- globin genes
results from deletions spanning both genes; the heterozygous
genotype is therefore written − −/αα. In populations where
specific deletions are particularly common, for example
Southeast Asia (SEA) or the Mediterranean region (MED),
it is useful to add the appropriate superscript as follows:–
SEA
–
/αα or– –
MED
/αα. It follows that when we speak of an
“α thalassemia determinant,” what we are referring to is a
haplotype; that is the state and function of both of the linked
α- globin genes together (αα).
α+ Thalassemia
There are two common forms of α+ thalassemia that are due
to the loss of one or other of the duplicated α- globin genes
from one allele, −α
3.7
and −α
4.2
, where 3.7 and 4.2indicate
the size of the deletions in kilobases. The α- globin genes are
situated in a highly homologous 4 kb duplicated region,
which is divided into three homology blocks (i.e. stretches of
DNA with highly similar sequence), X, Y, and Z, which are
themselves separated by non- homologous elements numbered I, II, and III (Figure1.4).
The most common deletion events arise from recombination between the X boxes or the Z boxes. Recombination
between the Z boxes, which are 3.7 kb apart is also known
as a rightward deletion and results in a thalassemic −α
allele and a relatively rare triplicated ααα
anti3.7
3.7
allele.
Similarly, recombination between the X boxes, which are
4.2 kb apart, known as leftward deletion, results in the −α
and ααα
α0 Thalassemia
anti4.2
alleles.
4.2
α0 denotes an allele that results in the complete loss of α-
globin expression from one allele. Most commonly, this is
due to the deletion of both α- globin genes from one allele
(− −). Over 50 different deletions removing both structural
α- globin genes from one allele have now been described
(Figure1.5).
These deletions can be grouped into those that are
restricted to the α- globin locus alone and those that aremuch
larger, removing flanking genes and in some cases extending
1–2megabases beyond the α- globin cluster (see ATR- 16 syn-
drome below). In contrast to deletions removing a single α
gene, these deletions are thought to occur as a result of illegitimate non- homologous recombination events. Deletion
8 Molecular Hematology
30–50 –10 0 10 20
(A)
(B) Rightward crossover
α
(C) Leftward crossover
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ψα
ψα
ψα
1
XYZXYZ
ψα
1
1
1
ψα
α
2
1
α
2
Inter-ζHVR
α
2
α
2
α
1
α
2
α
1
ψα2ψζ1 ψα1ζ2
α
1
α
1
α2α1θ
ααα
–α
ααα
–
1
3.7
4.2
α
anti
anti
1
3.7
4.2
3'HVR
Figure1.4 Mechanisms of the generation of the
common deletion forms of α+ thalassemia. (A) The
normal arrangement of the α- globin genes, with the
regions of homology X, Y, and Z. (B) The crossover that
generates the −α
generates the −α
Figure1.5 Some of the deletions that underlie α0 and
α+ thalassemias. The colored rectangles beneath the
α- globin gene cluster indicate the lengths of the
deletions. The unshaded regions indicate uncertainty
about the precise breakpoints. The three small deletions
at the bottom of the figure represent the common α+
thalassemia deletions. HVR, highly variable regions.
3.7
deletion. (C) The crossoverthat
4.2
deletion.
breakpoints may involve short sequences that are partially
homologous, such as the Alu- family of simple genomic
sequence repeats, which make up around 25% of the entire
sequence of the α- globin locus and are also commonly
found throughout the human genome. A variety of deletions
have been described, often named after the geographical
region where they have their highest frequency. Common
mutations like − −
genes but leave ζ- globin intact, whereas others remove both
ζ and α genes.
Larger deletions involving other genes have been observed
but only in the heterozygous state. Interestingly, while deletions within the α- globin locus can reach high frequencies in
certain populations, these very large deletions are never
found at polymorphic frequencies. This is probably due to
these mutations carrying a negative selective advantage due
to gene dosage effects, or unmasking mutations in the
retained homolog. In keeping with this, some individuals
with monosomy for a large segment of chromosome
16p13.3 have developmental abnormalities and cognitive
MED
and − −
SEA
remove both α- globin
impairment in addition to α thalassemia, a condition known
as ATR- 16 syndrome.
Non- deletional variants
Numerous single nucleotide and oligonucleotide mutations
within the α- globin structural genes have been reported that
either affect gene expression or protein stability. Mutations
may affect either the α2 gene (αTα) or the α1 gene (ααT) but
are most frequently found in α2. These mutations can involve
splice donor and acceptor sites such as α
nylation site mutations such as α
IVSI(−5nt)
AATAAG
and termination
2
α, polyade-
codon mutations such as the common Hb Constant Spring
variant that lead to extended unstable mRNA transcripts and
extended protein. Certain mutations within the coding
sequences themselves can give rise to hemoglobin variants
such as Hb Quong Zse, Hb Adana, and Hb Aghia Sophia that
are highly unstable, rapidly degraded and cause an α thalassemia phenotype even though strictly speaking, these are
structural variants.
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A comprehensive SNP and RNA analysis of samples from
Melanesian patients with α thalassemia who had intact
α- globin genes and enhancers revealed a point mutation
between the ζ and pseudo- ζ genes that creates a de novo
GATA1 binding site, and in so doing, forms a promoter
unit. Lying between the upstream enhancers and the α-
globin promoters, this new promoter is thought either to
compete with the native promoters for the activity associated with the enhancers or to act as an enhancer blocker
and thereby reduce α- globin transcription. Although rare
elsewhere in the world, this mutation is a relatively common form of α thalassemia in Melanesia, particularly in
Papua New Guinea.
Rare mutations causing α thalassemia
Some rare mutations causing α thalassemia have played key
roles in developing our understanding of how the globin
clusters are normally regulated. These mutations have mainly
been found in areas where thalassemia is otherwise rare. For
example, the upstream enhancers are vital for normal αglobin transcription and were initially identified by characterizing rare patients presenting with α thalassemia. To date,
over 25 patients have been described who have deletions of
the upstream elements while the α genes remain intact. Some
of these deletions may be very large; (αα)
CMO
extends to the
tip of chromosome 16p and involves repair of the end by
addition of telomeric repeats to non- telomeric DNA. In contrast, a recently reported deletion was as small as 742 bp.
Despite these variations, every reported deletion of this kind
removes MCS- R2, indicating its crucial role in regulating
human α- globin transcription.
A rare deletion (αα)ZF removes both α1 and θ and juxta-
poses the downstream gene (Luc7L) to the α2 gene. While the
α2 gene, its promoter elements, and the upstream enhancers
are intact, its expression is silenced by an anti- sense mRNA
from Luc7L transcribing through α2 and aberrantly methylating the CpG island associated with its promoter. This
mechanism is now known to underlie abnormal gene expression of other genes.
In addition to the patients described previously with
ATR- 16, ~200 male patients have been identified with
severedevelopmental delay, characteristic dysmorphic facial
features, and α thalassemia. This condition, ATR- X syndrome,
is caused by inherited mutations in the X- encoded chromatin
remodeling factor ATRX. This protein has been implicated
in various roles, including chromatin deposition, DNA
repair and telomere maintenance. Since its discovery in the
context of α thalassemia, ATRX has been identified as a
major tumor suppressor in up to 15% of cancers. The α
thalassemia phenotype is potentially explained by its role in
replication and DNA damage at the α- cluster, but this is still
under investigation.
Of interest, ATRX mutations are also found in a very rare
form of acquired α thalassemia known as ATMDS, which
most frequently presents in elderly (median age 68
years)
males in the context of myelodysplastic syndrome (MDS), a
clonal hematopoietic disorder. These cases are accompanied
by a variety of other mutations commonly seen in MDS.
Again, the mechanism causing α thalassemia is not fully
understood and under investigation.
β Thalassemia
Mutations resulting in quantitative reductions in β- globin
cause β thalassemia. Around 300 mutations have been
identified, which can be broadly described functionally.
Individuals with a β0 mutations are unable to produce any
β- globin from the affected allele, and β+ describe mutations
allowing some degree of expression. Most of these variants
involve point mutations or small insertions and deletions
involving the gene or flanking sequences. Mutations can
cause defects in transcription, RNA processing with or
without nonsense- mediated decay, and translational abnormalities including premature termination/protein truncation.
In contrast to α thalassemia, delsetions (~100 bp to 65 kb) of
the β genes are a relatively rare cause of β thalassemia. Some
large deletions also selectively involve the regulatory elements, and they all involve the enhancer element β- LCR2.
The molecular basis of β thalassemia often varies from one
population group to another suggesting that these mutations
have been independently selected during the recent evolution of Homo sapiens.
Defective β- globin gene transcription
A number of mutations cause β thalassemia by reducing the
amount of β- globin mRNA that is transcribed. These tend
to be point- mutations, predominantly in the promoter
region and 5′ UTR of the β- globin gene, especially involving the CACCC, CCAAT and TATA box sequences
(Figure1.6). The majority of these mutations result in mild
disease (β+ thalassemia), with β- globin transcription occurring at 10–25% of normal. The resulting phenotype can be
so mild that heterozygotes for some mutations, such as the
common Mediterranean −101 C > T mutation, have nearly
normal hematological indices and have been called “silent
carriers.”
Mutations causing abnormal processing of mRNA
Mutations disrupting the normal pattern of splicing to
remove the introns and generate a mature mRNA can cause
a variety of phenotypes, ranging from mild (β+) to severe (β0)
thalassemia. Mutations at the invariant splice site donor
dinucleotides, be they at the 5′ donor end (GT) or 3 acceptor
10 Molecular Hematology
Deletions
Point mutations
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IVS 2IVSI
PR C I FSNSSPL SPL FS
Figure1.6 The mutations of the β-globin gene that underlie β thalassemia. The heavy black lines indicate the length of the deletions. The point
mutations are designated as follows: PR, promoter; C, CAP site; I, initiation codon; FS, frameshift and nonsense mutations; SPL, splice mutations;
Poly A, poly A addition site mutations.
end (AG) completely disrupt splicing and result in β0 thalassemia, despite transcription itself being normal (Figure1.6).
Around these invariant donor and acceptor sequences are
the so- called splice site consensus sequences. These are the
SPL SPL FS
NS
sequence that is involved in RNA cleavage and polyadenylation. Transcripts can extend far beyond the normal PolyA
signal, with only 10% of all transcripts correctly processed
resulting in a β
32I
NS
+
phenotype.
Poly A
100 bp
last three bases of the exon and first six of the intron at the 5′
donor site and first base of the exon at the 3′ acceptor end.
An example is the common severe β+ IVS 1- 5 G > C mutation
that, like the other mutations of this type, also activates cryptic donor splice sites, thereby reducing the efficiency of normal splicing.
Gain- of- function cryptic splice sites also occur due to
mutations outside of the consensus sequences. These can be
generated by sequence variants in either introns or exons. A
prominent example of the latter is the generation of a cryptic
splice site that lies between codon 24 and 27 of exon 1 of
HBB, which functions as a donor as the variant generates a
GT dinucleotide. Splicing can occur from this site or from
the normal splice site at the exon junction. As many of the
mutations in the exons also change the encoded amino acids
in addition to the splicing defect, there may also be a variant
structural hemoglobin produced. These include Hb Malay,
Hb Knossos, but most prominently Hb E, which results from
a mutation of codon 26 (G > A) and is involved in approximately half of all severe β thalassemia worldwide. Mutations
in introns can also create aberrant splice sites. A notable
example that is commonly found in the Mediterranean area
is a G > A substitution at position 110in IVS- 1 of HBB. This
creates a new 3′ acceptor AG dinucleotide; approximately
90% of transcripts end up being spliced at this site, resulting
in a severe β+ thalassemia phenotype.
Finally, perturbations in posttranscriptional processing
can also result in β thalassemia. This class of mutations
generally involve the 5′ m7G CAP site, or the 3′ AATAAA
Mutations resulting in abnormal translation of
β- globinmRNA
There are two types of mutation in this class; those that disrupt the initiation codon, and those that result in the insertion of a premature stop codon (a “nonsense” mutation). The
first type generally involves substitutions in one of the three
bases in the ATG codon, but a 45 bp insertion disrupting the
initiation codon has also been described. Understandably, as
these mutations completely prevent translation, they have a
β0 phenotype. Rather more common are alleles resulting
from the generation of a premature termination codon. This
can happen through direct mutations producing a stop
codon such as the common Mediterranean codon 39 (CAG
to TAG) allele, which is the second most common β thalassemia mutation in the region. Alongside substitutions, insertions/deletions that are not three nucleotides long lead to
shifts in the reading frame and creation of a nonsense codon
further downstream. As translation from alleles carrying
these mutations is abolished, they also produce a β
0
phenotype. This is because while genes with nonsense mutations in
exons 1 and 2 such as these are transcribed normally, mutant
mRNA transcripts are not found in the cytoplasm (exon
3mutations are discussed below). This is due to a cellular
quality control mechanism known as nonsense- mediated
decay that clears mRNA transcripts containing premature
stop codons that otherwise would give rise to severely truncated proteins.
Beginnings: themolecular pathology ofhemoglobin 11
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Unstable β- globin chain variants
There are a set of mutations that give rise to unstable forms
of β- globin, sometimes so unstable that no protein can be
detected. An interesting group of these mutations are in
exon3. The mutant mRNAs are not cleared by nonsensemediated decay as the upstream exons are intact, and are
therefore translated into unstable variant β- globin chains.
These chains can precipitate in erythroid cells along with free
α- globin chains that are the hallmark of β thalassemia, result-
ing in a severe phenotype. Notably, this form of β thalassemia
is inherited in an autosomal dominant disorder, in contrast
to the vast majority of other forms, which require homozygous or compound heterozygous mutations.
Rare forms ofβ thalassemia
As for α thalassemias, there are rare variants in unlinked
trans- acting factors such as XPD, GATA1 and SUPT5H
that have been reported to cause mild forms of β thalassemia. Nevertheless, they may be associated with severe
abnormalities outside of the erythroid system. Although
these are of limited clinical importance with respect to
their phenotype of thalassemia, they highlight pathways
involved in globin gene regulation. At present is not clear
why mutations in general transcription factors should
have a differential effect on α- and β- globin expression.
While research into these disorders is still ongoing, it is
highly likely that a direct effect on transcription is the
mechanism underlying the erythroid phenotypes
observed.
δβ Thalassemia and hereditary persistence of
fetal hemoglobin (HPFH)
δβ Thalassemia encompasses a set of disorders with reduced
or absent δ- and β- globin production. There are two main
forms, δβ+ and δβ0 thalassemia. δβ+ thalassemia is caused by
the production of a δβ fusion gene, produced as a result of
unequal crossover between the δ and β genes during meiosis.
The 5′ end of this fusion gene includes the δ-
globin promoter, which is naturally a relatively ineffective promoter,
and so there is a reduced rate of synthesis of the newly
formed δβ fusion gene. The δβ fusion chains when combined
with α- globin form a new tetramer called Hb Lepore. In the
heterozygous state, individuals have the phenotype of β
thalassemia trait, and in the homozygous state, a moderately
severe anemia.
δβ0 Thalassemia on the other hand is caused by dele-
tions removing both δ- and β- globin genes, and may also
extend to involve the γA globin gene. There is a relatively
high level of Hb F production in these individuals, with
homozygotes producing only Hb F, and heterozygotes
producing 5–15% Hb F associated with thalassemic hematological indices.
Hereditary persistence of fetal hemoglobin (HPFH)
There are two classes of mutations that give rise to hereditary persistence of fetal hemoglobin (HPFH). The first is a
set of deletions (deletional HPFH) which remove the β-
globin genes themselves and/or regulatory elements that
control the γ- to β- globin switch. The second is a group of
single nucleotide or oligonucleotide mutations which affect
sequences within or close to the γ globin promoters: socalled non- deletional HPFH. The levels of Hb F in these
conditions can vary from <10% to 100% depending on the
HPFH sub- type.
Deletions within the β- globin locus sparing one or both
of the γ-
globin genes can lead to a raised Hb F in adult life.
Approximately 10 deletional forms have been described, all
removing the β- globin gene, and some also deleting δ-
globin (Figure 1.3B). Interestingly, deletions have been
described affecting an intergenic sequence near the ψβ
pseudogene (HBBP), which may play a role in γ- globin
silencing, possibly by segregating the fetal or adult genes
from the enhancers depending on developmental stage.
While several hypotheses have been proposed to explain
which deletions cause δ β0 thalassemia and which cause
HPFH, the underlying mechanisms have not been fully
explored at the present time.
The majority of mutations in the non- deletional forms of
HPFH lie within the γ- globin promoters (Figure 1.3A). A
range of small mutations has been described, from single base
substitutions to a 13 bp deletion, mostly in a region 100–200 bp
upstream of the transcriptional start site of both γ- globin
genes. These mutations can lead to an increased level of Hb F
ranging from 2% to 40%. The repressive proteins BCL11A and
ZBTB7A/LRF are known to bind to the −115 and −200 sites
respectively, and so mutations clustered around those sites disrupt binding and allow γ- globin transcription to occur. Other
mutations create binding sites for strong erythroid activators
such as GATA1 (−113 A > G), TAL1 (−175 T > C) and KLF1
(−198 T > C). Recent work has also shown that the −115muta-
tion, which disrupts BCL11A binding, leads to the recruitment of the transcriptional activator NF- Y to a nearby CCAAT
box, an example of transcription factor competition governing
stage- specific gene expression.
As previously mentioned, multiple trans- acting factors
have now been discovered that control γ- globin expression.
Patient studies have shown that naturally occurring variants
at or nearby some of these such as BCL11A, in the HBS1LMYB intergenic region and KLF1 among others are associated with elevated levels of Hb F. These tend to occur in the
context of a broader range of hematological abnormalities
alongside persistent γ- globin expression.
12 Molecular Hematology
)Hb Barts
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The pathophysiology andclinical
phenotypes ofthalassemia
Although the hemoglobinopathies are considered among the
best characterized of all monogenic disorders, as set out
above, the diversity and degrees to which α- and β- globin
expression are perturbed is very different from one molecular determinant to another. Nevertheless, it is possible to provide some guidance as to phenotype from the genotype. It is
likely that all of these variants have been selected for their
protection against falciparum malaria and consequently
many polymorphic α- and β- globin variants are found
together in tropical and subtropical regions of the world. In
addition, there are potentially many currently uncharacterized non- globin variants that influence the phenotypes of
patients with thalassemia. Even for these classical monogenic diseases, the complexity of the interactions is extensive. The hemoglobinopathies thus provide a sobering lesson
in molecular genetics for understanding more complex traits
and human diseases in which there may be hundreds of
genetic variants which interact to influence the phenotype
(e.g. type II diabetes, hypertension, asthma). Here, we summarize the common phenotypes seen in α and β thalassemia,
but emphasize that there may be considerable clinical variability within each group.
The α thalassemias
In α thalassemia, there is underproduction of the normal
α- globin chains, which contribute to fetal (α2γ2) and adult
(α2β2) hemoglobins. In fetal life, the excess γ- globin chains
form stable soluble tetramers (γ4) known as hemoglobin
Bart’s. Similarly, in adult life, the excess β- globin chains form
stable soluble tetramers, known as hemoglobin H (β4).
Neither hemoglobin Bart’s nor hemoglobin H form allosteric
tetramers and therefore do not effectively carry and release
oxygen; they are non- functional forms of hemoglobin. In the
common deletional forms of α thalassemia, erythroid precursors develop normally in the adult bone marrow, but
hemoglobin H does precipitate, leading to cell membrane
phospholipid dysfunction.
The consequent reduced erythrocyte deformability results
in extravascular hemolysis, particularly in the spleen. This
process tends to occur in aging red blood cells, especially in
times of physiological stress such as during infections.
Although this pathophysiology also applies to nonα thalassemia, depending on the particular mutation, this
may also be accompanied by some degree of dyserythropoiesis (Figure1.7).
The clinical phenotypes of individuals with α thalassemia
can be split into three broad categories: α thalassemia trait,
hemoglobin H disease and hemoglobin Bart’s Hydrops fetalis syndrome. α thalassemia trait is most commonly caused
deletional
γ Genes α Genes β Genes
γ mRNA α mRNA
γ globin
(γ4)
Fetal anemia
Figure1.7 The underlying pathophysiology of α thalassemia.
Inadequate production of α- globin leads to anemia and the production of tetramers of unpaired γ- globin chains in fetal life (Hb Barts, γ4)
and unpaired β- globin chains in adult life (HbH, β4).
α globin β globin
HbF HbA
β mRNA
Adult anemia
HbH (β
4
by single (−α/αα) or double deletions (− −/αα or −α/−α) of
the α- globin genes, and less commonly in those with a single
non- deletional variant (αTα/αα).
α Thalassemia trait is estimated to occur in ~5% of the
world’s population. Such carriers have a mild hypochromic
microcytic anemia and raised levels of hemoglobin Bart’s in
fetal and cord blood. In adults, rare cells containing hemoglobin H inclusions (1:1000–10 000) can be found in those
with the − −/αα genotype. These individuals are clinically
well, but their carrier status is of relevance for alerting clinicians that pregnancy screening may be required and also the
co- inheritance of α thalassemia may explain some unusually
mild forms of β thalassemia.
Hemoglobin H disease occurs in patients who inherit just
a single functional globin gene (− −/−α or − −/αT) or in
some cases homozygotes for a moderately severe nondeletional variant (α
T
α/αTα). Hemoglobin H disease is clinically variable but is usually associated with mild to moderately
severe anemia with some degree of jaundice and hepatosplenomegaly: the splenomegaly can be severe and cause hypersplenism. The majority of patients with the deletional forms
of hemoglobin H disease (− −/−α) do not require regular
blood transfusion. By contrast, those with non- deletional
forms of hemoglobin H disease quite often require blood
transfusion. This is particularly found in patients with hemoglobin H disease associated with the non- deletional chain
CS
termination mutant hemoglobin Constant Spring (−/α
α).
Non- deletional alleles (αTα) usually produce less α- globin
that the common deletional alleles (−α) because they
affectthe α2 gene, which is more highly expressed than the
α1 gene. Patients with either deletional or non- deletional
hemoglobin H disease may have episodes of hemolytic
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