Download Hemoglobin A2: origin, evolution, and aftermath

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The Journal of
The American SocieQ of Hematology
BLOOD
NOVEMBER 1, 1991
VOL 78, NO 9
REVIEW ARTICLE
Hemoglobin A,: Origin, Evolution, and Aftermath
By Martin H. Steinberg and Junius G. Adams 111
T
H E MELANGE of hemoglobins present in the erythrocytes of humans includes hemoglobin A, (HbA,),’., a
Its unique
tetramer of a- and &globin chains (a26,).
characteristics reside in the n~n-a-chain.~
HbA, is physiologically unimportant because it is normally less than 3% of
the total Hb. The study of HbAz has provided insights into
the evolution and phylogeny of globin genes and enhanced
our understanding of gene expression and globin synthesis.
HbA, has substantial clinical relevance. Its concentration
fluctuates in the thalassemia syndromes and some acquired
diseases, so that its measurement provides a useful diagnostic aid. This review will focus on the structure, function, and
synthesis of the &-globin chain and HbA,, as well as the
features of this Hb that give it clinical utility.
CHARACTERISTICS OF THE &GLOBIN GENE
The evolution of the &globin gene was initially very
confusing, because HbA, was present in humans, apes, and
New World monkeys, but not in Old World monkeys.34”
This finding was seemingly at variance with the evolutionary
data which indicated that humans and Old World monkeys
diverged after the divergence from New World Monkeys.
This conundrum was ultimately solved with the finding that
&globin genes are indeed present in Old World monkeys,
but have been inactivated by mutation.3842
After examination of the gene sequence of a number of
primate species, the origin of the &globin gene was first
thought to have occurred relatively recently (about 40
nlillion years
However, studies of the globin
genes of mice, rabbits, and other primates make it more
likely that this divergence occurred before the mammalian
radiation approximately 85 to 100 Mybp, at about the same
time as the E- and y-globin genes diverged.M4nIt is clear
from the comparisons of &globin genes among mammals
that the &globin locus has not evolved as an independent
lineage, but has evolved in concert with the p-globin gene.
In each of the mammalian orders examined to date, the
&globin locus has acquired characteristics of the P-globin
locus through gene conversion (a nonreciprocal exchange
of genetic material between the two linked homologous
genes).” 44 4‘47 cu These gene conversions have most often
occurred in the coding regions, rendering these regions
useless in the quest for the primordial %globin gene. Thus,
the evolutionary origin of the &globin gene has been
performed using flanking and intervening sequence data
(especially I V S I I ) . ~ ~
Gene conversion and the &globin gene. The nonallelic
gene conversion events postulated to occur during the
evolution of the &globin gene are thought to have been rare
when compared with the gene conversion between the two
y-globin loci.44However, Petes” has made an interesting
observation concerning the structural variants of HbA,.
Linkage relationships and chromosomal location of the
&globin gene. It has long been known that the p- and
&globin genes were closely linked. These initial linkage
data were derived from the study of families in which both
p- and &globin variants were ~egregating.~.“’
The location of
the p-globin gene family on the short arm of chromosome
11 involved a variety of molecular techniques.”.” The linear
arrangement of the @-globingene cluster was determined
from data derived primarily from gene mapping and is
shown in Fig 1.”’-*‘
These genes have also been completely
sequenced.”-” The general form of the &gene is akin to all
other globin genes with three coding regions (exons) and
two intervening sequences (introns).
Evolution of the &globin gene. Because of the large body
of sequence data that is available from the globin genes as
well as the proteins that they produce, this gene family has
provided an excellent opportunity to examine its molecular
evolution. The globin genes are all thought to have arisen
from a common globinlike heme protein (Fig 2). The
earliest duplication of this ancestral gene led to the divergence of myoglobin and the globins that comprise Hb and
most likely occurred approximately 700 million years before
From the Department of Medicine, University of Mississippi School
present (Mybp).32 The next duplication gave rise to the
ofMedicine, and VA Medical Center, Jackson, MS.
divergence of the a-globin genes and occurred approxiSubmitted June 17, 1991; acceptedAugust 21, 1991.
mately 450 Mybp.32The P-globin gene is thought to have
Supported by Research Funds of the Department of VeteransAffairs.
duplicated about 180 to 200 Mybp into an ancestral gene for
Address reprint requests to Martin H. Steinbe%, MD, (151), VA
E- and y-globin and an ancestral gene for 6- and P-gl~bin.’~ Medical Center, 1500 E Woodrow Wilson Dr, Jackson, MS 39216.
Approximately 110 to 130 Mybp, the E / ? parent gene
This is a US government work. There are no restrictions on its use.
diverged to establish the E and y lineages.
0006-4971191 17809-0049$0.00/0
Blood, Vol78, No 9 (November 1). 1991: pp 2165-2177
2165
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2166
STEINBERG AND ADAMS
Chromosome 11
H
5kb
Fig 1. The arrangement of the plike globin gene cluster.
-
Two of the then-described 10 &-globinvariants could have
arisen by gene conversion. The &globin variants HbA,
Flatbush (622 ala + glu) and HbA, Coburg (6116 arg his)
both contain a P-globin amino acid in 1of the 10 amino acid
residues where these two globins differ. The occurrence of
these variants is much greater than would be expected for
random mutation. Therefore, Petes suggested that these
two variants could represent gene conversion events. Another variant that was found subsequent to Petes’ hypothesis, Hb Parchman (622 ala + glu, 50 ser thr), could also
be due to gene conversion rather than to a double crossover
as proposed by the investigators.s2This hypothesis is easily
testable in the variant HbA, Coburg, because a gene
conversion event would result in a codon 116 change of
CGC to CAT, while a point mutation would result in a CGC
to CAC change.’’
Synthesis of the &globin chain. Clearly gene conversion
has maintained strong sequence homology between the
human P- and &globin genes. This homology is especially
striking in the coding regions where there are 10 amino acid
differences and 31 nucleotide differences. However, the
quantitative expression of these two genes is strikingly
different. HbA makes up more than 95% of the adult
hemolysate, while HbA, comprises only 2% to 3%. The
molecular stability of these two molecules appears to be
almost identical, making it highly unlikely that difference in
posttranslational survival of the two molecules accounts for
the low proportion of HbA,. In studies where reticulocytes
were incubated with radioactive precursor amino acids, it
became apparent that the &globin chain is synthesized at a
reduced rate in the bone marrow and not at all in reticulocyte~.’~.’~
These experiments were extended by fractionating
bone marrow erythroblasts into fractions of different levels
P
My b.p.
I
A
-190
AA
e
Y 6
p
-130
c*
85 (Mammalianradiation)
Fig 2. The evolutionary history of the &-globingene.
of maturity. These experiments showed that there was a
progressive decrease in &-globin synthesis in relation to
P-globin in increasingly mature cells. A relative instability
of 8-globin mRNA was proposed as a mechanism for the
premature decrease in &globin synthesis? Using highly
selective probes for P- and &globin mRNA, it was found
that the half-life of &globin mRNA was less than one third
that of P-globin mRNA, supporting this hypothesis.”
Despite the strong evidence that 6-globin synthesis decreases during the maturation of erythroid precursors, this
decrease does not account for the great discrepancy between P- and &globin synthesis. In normal bone marrow
cells, the synthesis of &globin chains is less than 2% of total
non-a-globin synthesis. In addition, even in the youngest
fraction of bone marrow erythroid cells examined, the
6:P-globin synthesis ratio did not exceed the usual HbA2:
HbA ratio found in the peripheral blood. Furthermore, the
translation rates of the two globins were the ~ a m e . ~ ‘
These findings strongly suggested that the rate of transcription of S-globin gene must be less than that of the
P-globin gene. When the in vitro transcription of non-aglobin genes was compared, it was found that P-, E-, and
y-globin genes are transcribed with equal efficiencies, but
that the transcription of the 6-globin gene is far less
efficient.” Humphries et a P compared the expression of a-,
P-, and &globin genes in monkey kidney cells. Under
conditions that promoted optimal transcription of each
globin gene in this system, &globin gene transcription was
found to be 50 times less efficient than that of the P-globin
gene. This transcriptional deficiency of the &globin gene
approximates the synthesis of &globin in normal erythroid
cells. Humphries et al also made hybrid constructs of the 6and P-globin genes. When the 5‘ end of the &globin gene
was replaced by the homologous portion of the P-globin
gene, transcription of this hybrid gene was equal to that of
the normal P-globin gene. However, when the 5’ portion of
the p-globin gene was replaced by the homologous portion
of the &globin gene, the transcription of the hybrid gene
was like that of the &globin gene. These findings suggested
that sequences in the 8-globin gene promoter were responsible for the decreased transcription of this gene. One of
the most striking differences between the P- and &globin
genes is that the usually conserved CCAAT box is the 5’
promoter region is CCAAC in the &globin gene and is the
expected CCAAT in the p-globin gene. Because the CCAAT
box is required for normal transcription of the rabbit
P-globin gene? this difference is thought to account for
most of the differences in the synthesis of P- and S-globin
synthesis. It has been shown that IVS-I1 of the &globin
gene also acts to reduce its synthesis by mechanisms that
are unclear now.M’
HbA, is synthesized in all erythroid progenitors and
therefore its distribution in the blood is pancellular.61This
contrasts with the expression of the y-globin gene in adults,
where only a minor fraction of erythrocytes (F cells)
contains HbF. This difference in the expression of these two
minor Hbs found in adults may result from the restriction of
y-gene expression to a very few erythroid precursors. The
special mechanisms that govern switching from fetal to
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HEMOGLOBIN A,
2167
Lepore globin genes produces a chromosome with a 8-PS-P
adult Hbs within the mammalian @-globin gene cluster
configuration. Two of these “anti-Lepore” variants have
inactivates the y-globin genes as 6- and P-gene transcripbeen found, Hb Miyada” and Hb P N i l o t i ~ . ~Because
~.~~
tion is activated.62
these individuals have a normal P-globin gene, they do not
Function of HbA, and the &globin chain. HbA, has
exhibit a thalassemic phenotype. The anti-Lepore globin
functional properties that are nearly identical to those of
gene has the P-globin gene promoter, so synthesis of these
HbA. It has similar oxygen affinity, Bohr effect, and
globins would be expected to be near that of the P-globin
~ooperativity.6~,~~
Its response to 2,3 bisphosphoglycerate is
gene.
In Hb P Nilotic heterozygotes, the variant Hb was
also similar to that of HbA.65The thermal stability is greater
This
than that of HbA.& HbA, inhibits polymerization of HbS.67 found to comprise 21% to 28% of the hem~lysate.~~.~’
proportion closely approximates that expected if the P
The residues 622 (Ala) and 687 (Gln) appear to be the
Nilotic globin chain was synthesized at nearly the same rate
important inhibitory sites. In instances where the HbA, is
as the normal P-globin chain. It has been shown that the
exceptionally high, and in the presence of elevated HbF
synthesis
of Hb P Nilotic decreases in reticulocytes as
levels, the combination of these two Hbs may modulate the
compared with bone marrow cells,77but this decrease in
phenotype of HbS-Po-thalassemia.68The positive charge of
synthesis is not a major determinant of the total synthesis of
HbA, may endow it with properties similar to other posithis globin because it appears to accumulate in the expected
tively charged Hbs, such as HbC, relative to its interaction
amounts in red blood cell (RBC) precursors. The synthesis
with the erythrocyte
HbA, has a higher affinity
of
the PG-globin chain of Hb Miyada is apparently synthefor erythrocyte membrane band 3 than does HbA.70While
sized somewhat less efficiently than Hb P Nilotic, compristhe interaction of HbC with the membrane is believed to
ing only 17% of the hemolysate in heterozygous individudetermine the pathophysiologic properties of HbAC and
a l ~The
. ~ synthesis
~
of this abnormal globin was also found
HbCC cells,71the concentrations of HbA, make it doubtful
to
be
greatly
decreased
in reticulocytes when compared
that it can meaningfully affect cation transport and mean
with
bone
marrow
cells.78
corpuscular Hb concentration.
The non-a-globin chain of Hb Parchman is also informaHybrids of the p and &globingenes. The Lepore Hbs are
tive in this regard. If Hb Parchman arose a double crossover
the products of SP hybrid globin genes and have provided
as initially suggested, it should contain the &globin gene
insight into the mechanisms of decreased &globin synthepromoter,
IVS-I from the p-globin gene, and IVS-I1 from
sis. In 1958, Gerald and Diamond7* showed that the inthe &globin gene. The non-cu-globin chain of Hb Parchman
dividuals who carried this variant exhibited hematologic
is synthesized at the same rate as the normal &globin chain,
manifestations identical to P-thalassemia, but had an elecsupporting the suggestion of Kosche et @
aI’ that the detrophoretically slow-moving Hb that comprised 10% to
creased
synthesis
of
the
&globin
chain
is due to the
15% of the total hemolysate. Subsequently, B a g l i ~ n i ~ ~
presence
of
the
CCAAC
promoter
and
the
&globin gene
showed that the non-cu-globin chain of Hb Lepore had
IVS-11.
&globin sequences at its amino terminus and P-globin
&Globin variants. The known structural variants of the
sequences at its carboxyl terminus. The Lepore Hbs most
&globin
gene are depicted in Table 1. Although these
likely arose from a nonhomologous crossing over event in
variants
may
be unstable or have elevated oxygen affinity,
which the P-globin gene from one chromosome mispaired
they are not associated with a clinically significant phenowith the &globin gene of the other chromosome during
type because, as mentioned previously, HbAz has no detectsynapsis. The result of this recombination is the deletion of
able
effect on the oxygen transport of the RBC due to its
a segment of DNA approximately 7 kb in length from a
low
proportion.
Because HbA, variants have no effect on
point within the transcribed portion of the &globin gene to
phenotype,
the
number
of structural variants of the &globin
a corresponding point in the P-globin gene. Therefore, the
Lepore chromosome does not have a normal 6- or @-globin gene is far less than that of the a-or P-globin gene.
gene.
CLINICAL FEATURES OF HBA,
Current concepts of how the Lepore SP fusion gene
variants produce the phenotype of thalassemia are that the
Measurement of HbA,. The &chain of HbA, contains
&globin gene sequences located at the 5’ portion of these
two additional positive charges compared with the P-chain
fusion genes result in their characteristically reduced rate
of HbA. This facilitates its separation by electrophoretic
and chromatographic methods that rely on charge differof ~ynthesis.’~The major difference in this region, as
mentioned previously, is that in the &globin gene, the
ences to resolve proteins from one another. Hb electroCCAAT box promoter sequence is CCAAC. Another
phoresis on starch or polyacrylamide gels or cellulose
puzzling feature of the Lepore globin chain was that is was
acetate membranes affords wide separation of HbA, from
HbA and HbF and a means to quantify accurately this
synthesized at a higher rate than the &globin chain of
minor Hb ~ o m p o n e n t . ~ Acid
HbA,. This increased synthesis of the hybrid globin is most
~ . ’ ~ agar gel electrophoresis
likely due to the presence of the second intervening
does not resolve HbA, from HbA. The ease of working with
sequence of the P-globin gene.@The rare homozygous forms
cellulose acetate membranes, their commercial availability
of Hb Lepore, as well as combination of Hb Jipore with other
and reasonable cost, makes this the current electrophoretic
P-thalassemia genes, are usually expressed clinically as modermethod of
The low level of HbA, in erythrocytes,
ately severe forms of thalassemia major.74
and the narrow range separating normal from abnormal,
The reciprocal product of the crossover that produces the
causes methods of measurement that are facile from the
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2168
STEINBERG AND ADAMS
Table 1. Structural Variants of the &Globin Gene
Mutation
GIG + CCG
CAT -+ CGT
A A I + nqc! or AAG
GGC + CGC
CIG + GAG
GCA -+ G F
-
Amino Acid
Residue No.
Abnormality
1 (NA1)
2 (NA2)
Val + Ala
His + Arg
A, Niigata
A, Sphakia
12 (As)
16(A13)
20 (82)
22 (84)
Asn + Lys
Gly+Arg
Val -+ Glu
Ala + Glu
A, NYU
A;
A2Roosevelt
A, Flatbush
Hb Name
24 (B6)
25 (87)
43 (CD2)
47 (CD6)
51 (D2)
69 (E13)
Gly + Asp
Gly + Asp
Glu + Lys
Asp + Val
Pro + Arg
Gly + Arg
A,
A,
A,
A,
A,
A,
Victoria
Yokoshima
Melbourne
Parkville
Adria
Indonesia
GAG -+ GIG
90 (F6)
98 (FG5)
99 (Gl)
116 (G18)
116 (G18)
121 (GH4)
125 (H3)
136 (H14)
142 (H20)
Glu -+ Val
Val + Met
Asp + Asn
Arg -+ His
Arg + Cys
Glu -+Val
Gln + Glu
Gly -+ Asp
Ala + Asp
A,
A,
A,
A,
A,
A,
A,
A,
A,
Honai
Wrens’
Canadat
Coburg
Corfu
ManzanaresI
Zagreb
Babinga
Fitzroy
-+
CGC + CAC or CATS
+ JGC
G&I + G I A
CAA + GAA
GGT + GAT
GCT + GAT
-CGC
% Variant
Reference
XR
XR
-
184
185
Indian
Eastern European
Black American
Iraqi
Black American
Babinga pygmies
Iraqi
Japanese
Italian
?
Italian
Malayan
Sumatran
Japanese
Black American
East Indian
Italian
Greek
Spanish
Yugoslavian
Babinga pygmies
Greek
q = .004
R
q = .009
XR
1.o
1.o
1.o
186
6.7.10
187
188
1.3
1.6
1.4
189
190
191
192
193
194,195
0.8
0.2
1.8
1.4
0.5-1.5
0.4
1.1
1.o
1.4
196
197
198
199
204
200
20 1
202
203
Population
GGT -+ GAT
GGT + GAT
GAG +@
-G
GAT + G F
CCT -+ CGT
GGT + CGT
GTG ATG
GAT + &IT
Frequency
Japanese
Cretan Canadian
R
q = ,024
XR
XR
XR
XR
XR
q = .001
q = .024
XR
XR
XR
XR
R
XR
XR
q = ,0007
XR
-
44% of HbA,
1.2
-
Abbreviations: R, rare; XR, extremely rare.
*Unstable.
tHigh oxygen affinity.
*See text for details concerning possible gene conversion.
standpoint of the clinical laboratory, such as densitometric
tracings of electropherograms, to be i n a c c ~ r a t e . ~For
~-~
acceptable accuracy, the HbA, fraction must be eluted and
measured spectrophotometrically.84Refrigeration and freezing may reduce the HbAz percentage in stored hemolysates.” The differential elution of HbA, in minicolumns is
reliable, rapid, and inexpensive.’””
Both electrophoresis and conventional methods of column chromatography are incapable of separating HbAz
from Hb variants that contain similar charge differences.
Unfortunately, the very common HbC and HbE are in this
group of positively charged Hbs. In the company of abnormal globins that contain only a single additional positive
charge, like the sickle p-chain, the HbAz level has been
reported to be higher than normal.%However, good laboratory technique and the choice of appropriate separative
methods can circumvent this complication and permit the
use of electrophoresis and chromatography to measure
HbA, in the presence of HbS.80,95,97,98
High performance liquid chromatography (HPLC) can
separate HbA, from other Hb types as well as discriminate
the 8-globin chain of HbA, from a,p, and y-globin chains.
Cation exchange columns afford excellent resolution of
HbA, from HbS and HbC,99-1”1
but the time and expense of
this method detract from its clinical use. The quantification
of non-cu-globin chains using a C, column (Vydac, Hisperia,
CA) provides a useful surrogate for the measurement of
intact Hbs and contributes an effective way of assessing
HbA, in the presence of HbC or HbE.’02-1”
HbA, may be measured i m m u n o l o g i ~ a l l y . This
~ ~ ~ ~tech’~~
nique has the virtue of specificity. The levels obtained
correlate well with the more traditional methods of measurement. To date, this procedure has not enjoyed wide clinical
application.
HbA, in health and disease. There is little &-chain
synthesis in utero and the accumulation of HbAz does not
become easily measurable until late in gestation. The HbA,
level in normal newborns is 0.27% f 0.02%.” The amounts
of HbAz vary with gestational age; they are lowest in the
least mature infants.”’ HbA, levels do not increase synchronously with HbA, but lag behind; the HbA/HbA, ratio is
about 100 at 32 weeks of gestation and 75 at 45 weeks.”’
The “adult” HbA/HbA, ratio of about 40:l is not reached
until at least 6 months of age. This sluggish response of
HbA, during maturation may reduce its value for the
diagnosis of P-thalassemia in young infants.”’ The stable,
“adult” level of HbA, is 2.5% to 3.5%. In the presence of an
a-globin variant, such as HbGPh“ade’ph’a
, the variant a-chain
combines with the 8-chain to form an Hb tetramer with the
structure, aVar’an128T11’
This tetramer, often called HbG,,
usually comprises less than one half the total amount of
HbA,. The HbA, “variant,” HbG,, is more positively
charged than HbA, because of the positive charge of the‘a
chain, and is easily separated from HbA,. Extra HbA, bands
are a valuable clue to the presence of variant a-globin
chains, although, depending on the charge of the a-variant,
they may or may not separate from the major Hb bands.”’
Glycosylated forms of HbA,, analogous to the minor
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2169
HEMOGLOBIN A,
components of HbA, are present and can be quantified by
HPLC and isoelectric f o c ~ s i n g . " ~As
~ " ~with HbA,,, these
glycohemoglobins are elevated in poorly controlled diabetics.
Low HbA,. The causes of reduced levels of HbA, are
shown in Table 2. Other than the age-related decrement
from "normal" found in infants and very young children,
low HbA, values are in most instances the result of either
reduced synthesis of the &globin chain (thalassemia), or
posttranslational modifications in the assembly of the HbA,
tetramer (Table 2). Reduced HbA, tetramer assembly can
result from either acquired or genetic disorders. In either
case, the proximate cause is the same; a reduction in the
synthesis of a-globin chains.
Posttranslational causes of reduced HbA,. Hb tetramer
assembly follows rapidly on the formation of dimers consisting of a- and non-a-globin chain^.''^^"^ Dimer formation, in
turn, is dependent on the charge of the non-a-chain.
Normal a- and P-monomers have nearly equivalent positive
and negative charges, respectively, and are united by
electrostatic attraction. The &-chain is more positively
charged than the p-chain (or y-chain). Under normal
conditions when there is a-chain sufficiency or slight excess,
HbA is formed in priority to HbA,, because a p dimers form
in preference to a8 dimers. When the supply of a-globin
chain is limited, the effect of charge is exaggerated, as the
p-chain (and y-chain) compete more effectively than the
&chain, for the limited quantity of a-globin."
Acquired conditions causing low HbA,. A number of
acquired conditions are capable of reducing a-globin synthesis relative to that of non-a-chains. Most seem to have their
effect through the common mechanism of absolute or
functional iron deficiency. In the absence of sufficient iron,
a repressor of initiation of protein synthesis is formed."'
This may preferentially affect a-, rather than non-a-globin
chain initiation, resulting in a relative deficiency of a-chains.
Patients with iron deficiency anemia have reduced levels of
HbA,. This is most apparent in individuals with the most
severe iron deficiency."'-'22 Individuals with anemia, microcytosis, and low levels of HbA, on the basis of iron
deficiency might be mistaken for carriers of one of the more
severe forms of a-thalassemia. When iron deficiency and
Table 2. Causes of Reduced Levels of HbA,
Reduced &Globin Synthesis
Neonatal period
8-Thalassemias
8p and yap-Thalassemias
Lepore Hbs
&Globin chain variants
Reduced HbA, Tetramer Assembly
Genetic
a-Thalassemia
Acquired
Iron deficiency
Lead poisoning
Sideroblastic anemia
Myeloproliferative disorders
p-thalassemia coexist, the HbA, level has been reported to
decrease,"' although it may remain within the range expected for thalassemia heterozygotes.l' Iron deficiency may
not affect the HbA, in all patients with p-thalassemia. We
recently studied two patients heterozygous for the -88
C + T p'-thalassemia who also had iron deficiency anemia.
Their HbA, levels were appropriately elevated while they
were iron deficient and did not change during iron repletion. The iron utilization defect associated with sideroblastic anemias may also reduce HbA, level^."^
There may be profound effects on a-globin synthesis in
certain myeloproliferative disorders. The expression of all
a-globin genes is affected and the phenotype can mimic the
genetically determined HbH disease. While these instances
are uncommon, they can be associated with low HbA,
values. The HbA, level in acute myeloid leukemias (AML)
is lower than in acute lymphocytic leukemias.IzsThis, and
other hematologic differences between these groups, suggests that the AML clone involves the erythroid lineage. In
juvenile chronic granulocytic leukemia a pattern very similar to fetal erythropoiesis may develop. Fetal Hb levels may
soar, accompanied by low HbA, values, recapitulating
normal neonatal findings and the reciprocity of y- and
&globin gene expression.126-128
Erythroleukemia has also
been associated with very low levels of HbA, in the absence
of HbH.'29,'M
Conceptually, these acquired "a-thalassemias"
bridge the difference between the reduced HbA, secondary
to acquired disease and low HbA, associated with genetic
abnormalities of a-globin synthesis.
Genetic conditions associated with low HbA, a-Thalassemia is extraordinarily common in certain populations.
The deficit in a-chain synthesis ranges from trivial to
extreme and the level of HbA, varies commensurately with
the deficit in a-globin synthesis.'" With the mildest types of
a-thalassemia, HbA, values in individuals may be indistinguishable from normal. When a-globin production is impaired significantly, the reduction in HbA, is dramatic. In
21 patients with HbH disease, the HbA, ranged from 0.5 to
1.8% and averaged 0.8%.13, Homotetramers of &chains
have been reported in a-thalassemia hydrops fetalis.
The Sthalassemias. Thalassemia-inducing mutations
may affect the &globin gene. Uncomplicated &thalassemia
has no clinical repercussions. In &+-thalassemia,both heterozygotes and homozygotes have reduced HbA, levels.
When the &gene is totally inactivated (F-thalassemia), the
heterozygote has half normal HbA, levels, while HbA, is
absent in the homozyg~te."~
When &gene expression is abolished as a result of large
DNA deletions that remove the p-, and at times the
y-globin genes, as well as the &gene, the resulting phenotype is a consequence of impaired p- or y-gene expression.
Heterozygotes for GP-thalassemia and gene deletion hereditary persistence of fetal Hb (HPFH), have half-normal
HbA, levels; homozygotes have no HbA,.'I9 With Lepore
Hbs there is a 50% reduction of HbA, level in heterozygotes
and no HbA, in homozygotes. While the percentage of
HbA, may be low when &globin gene expression is abolished, the absolute level, expressed as picograms of HbA,
per cell, is slightly elevated. This reflects increased synthesis
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2170
STEINBERG AND ADAMS
of HbA,from the chromosome in trans to the gene deletion
(see below).
Early descriptions of &thalassemia were based on hematologic and family studies. In the context of our present
ability to define the thalassemias at the molecular level,
some of these reports are unreliable. The mutations described in the 6-thalassemias are shown in Table 3. There
have been a few surprises among these mutations and they
resemble the defects that have been found to cause other
thalassemias like frameshift mutations, splicing defects,
gene deletions, and possible unstable H ~ s ’ ” . ”(~Trifillis P,
Ioannou P, Schwartz E, Surrey S: Identification of four
novel &globin gene mutations in Greek Cypriots using PCR
and automated fluorescence-based DNA sequence analysis. Blood [in press]). Several of these &thalassemiacausing mutations are also &globin structural variants.
These variants are similar to the thalassemic hemoglobinopathies caused by some a- and P-globin gene mutants.
Reflecting the relative rarity of gene deletion with the
p-like globin gene cluster, there is but a single reported
example of gene deletion causing “pure” &thalassemia. A
7.2-kb deletion removed most of the &gene and stopped
just 3’ to the +P-gene. While the initial molecular characterization of this deletion suggested that it inactivates the
P-gene, subsequent studies make it likely that a p+thalassemia mutation was responsible for the reduced
expression of the P-gene in cis to this deletion.’3s3139
The
6-thalassemias have been reported most often in Japanese,
Italian, and Greek populations. Whether this represents
their true distributions is not known, as extensive surveys
for these barely detectable conditions have yet to be
reported. In Italians, and potentially in other ethnic groups
where P-thalassemia is common, the coexistence of
&-thalassemiamay cause the HbA, to be normal in the presence
of P-thalassemia.I3l Interactions between a-thalassemia and
p-thaIassemiamay also result in normal HbA, vah~es.’~’
These
“normal HbA, p-thalassemias” may escape detection if
diagnosis depends on the measurement of HbA, levels
alone.
Table 3. Molecular Causes of the 8-Thalassemias
8”-Thalassemia
Codon 91 frameshift, insertion of A; premature stop at position
94135
T + C, IVS-1, position 1. Abolishes splicing’”
T + C, position -77. ?Transcriptional defect’=
T + C, position 2 of condon 141 (leu-pro). ? Unstable Hb*
A + G, IVS-2 3’ splice site. Normal splicing disrupted*
AAG + AG, codon 59. Frameshift; premature stop at codon
~~137.13111
7.2-kb deletion, extending from 3’ 6p through IVS-2 of the
8-gene.”.lm
8’-Thalassemia
G + T, position 1 of codon 27 (ala-ser). 7 Cryptic splice site activation’”
C + T, position 1 of codon 116 (arg+cys).*
‘Trifillis P, loannou P, Schwartz E, Surrey S: Identification of four
novel &-globin gene mutations in Greek Cypriots using PCR and automated fluorescence-based DNA sequence analysis. Blood (in press).
A priori, mutation in the &globin gene should be as
frequent as that in the P-globin gene. Yet, far fewer
6-thalassemia-causing mutations and &globin variants have
been described. The explanations for this anomaly are
probably twofold. First, the lack of clinical or hematologic
abnormalities associated with &thalassemia (or &chain
hemoglobinopathies) makes its detection difficult; second,
the inconsequential hematologic change of &thalassemia
provides an insufficient basis for natural selection to protect
the carrier from Falcipamm malaria.
HbA, levels of approximately half normal are present in
individuals heterozygous for &-chain v a r i a n t ~ . ’ ~In~ ’this
~~
instance, the level of the HbA, variant is equivalent to
HbA,, and the sum of both minor Hbs is equal to the
normal HbA, level. Homozygotes for HbA,variants have no
normal HbA,.
High HbA,. High HbA, levels are a result of
P-thalassemia in almost all instances.’ With few exceptions,
an elevated level of HbA,,in the presence of microcytic
erythrocytes, equates to the diagnosis of heterozygous
P-thalassemia. Borderline levels of HbA, are not common
when modern methods of measurement are used. In these
instances, the absence of microcytosis makes the diagnosis
of heterozygous P-thalassemia unlikely, although with some
of the “mild” P-thalassemia mutations the erythrocytes may
be
However, P-thalassemia may be present
with normal levels of HbA,. One important caveat is the
case where the coexistence of &thalassemia or a-thalassemia
conspires to reduce the HbA, levels toward normal. The
HbA, level may be higher in heterozygous v-thalassemia
than p+-thalassemia.I3’This is likely to be a result of the
greater impairment of p-chain synthesis in v-thalassemia.
The overlap in values precludes any diagnostic utility of this
observation. Only a minority of the point mutations causing
P-thalassemia are not associated with a raised HbA, level.
It appears that p-thalassemia mutations that lead to only
mild impairment of P-globin synthesis are accompanied by
more modest elevations of HbA, than the more severe
p-thalassemia-causing mutation^.'^'^' This may be a result
of posttranslational dimer assembly, where a relatively
nominal suppression of P-chain synthesis and minor excess
of a-globin chains leads to less ab-dimer formation. An
exception may occur when the mutation affects the P-gene
promoter. Codrington et all” propose that point mutations
in the P-globin gene promoter may alter its binding of
transcription factors and augment &gene transcription in
cis. In one patient with the -88 C + T p’-thalassemia and
HbA;in trans, the total of both HbA, types was about 7%.
They also reported an HbAzlevel of 5.4% f 0.4% (4.5% to
6.6%) in 10 heterozygotes with this same mutation. We
found an average HbA, of 4.87% (4.1% to 5.2%) in six
heterozygotes with this mutation from a single family
(unpublished data, July 1991).There appears at this time to
be insufficient data to precisely define the effects of p-gene
promoter point mutations on HbA, synthesis.
HbA, levels in homozygous p-thalassemia are variable
and of little diagnostic value. This is a result of the striking
increases of HbF that typify the severe P-thalassemias.
Transcription of the &globin gene appears to vary in a
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HEMOGLOBIN A,
reciprocal fashion with that of the y-globin gene. This
reciprocity is evident as HbF levels decrease rapidly during
the last trimester of gestation and is also observed in the
P-thalassemia syndromes.'" In homozygous P-thalassemia,
cells with the highest HbF levels have the lowest HbA,
concentrations.Ia The relationship between HbA, and HbF
was strikingly illustrated in a patient with p-thalassemia
trait and the Swiss type HPFH receiving chemotherapy.
The HbF level increased dramatically, from 4.5% to 26%,
accompanied by a decrease in HbA, from 4.5% to 2.4%.'49
The cause of increased HbA, in heterozygous @-thalassemia
appears to reside at both the transcriptional and posttranslational level of Hb
There is an increase in both
the percentage and absolute amount of HbA, present, with
the former about twice as great as the latter. Reduced
production of P-globin, with a relative excess of a-globin
chains, favors the formation of a 8 dimers and the assembly
of HbA, tetramers. If part of the cause of elevated HbAz
was due to posttranslation perturbations, the product of
each &-globin gene should contribute equally to this increase; ie, the effect should be present both in cis and in
trans to the @-thalassemia gene. This has been shown
directly in the study of families where a structural variant of
the &globin chain segregates independently from the
P-thalassemia-causing m u t a t i ~ n . ' ~ ~Increased
~ ~ ~ . ' ~ ' 6-gene
transcription, as a result of a p-thalassemia-causing mutation, might be expected to occur only in cis. The mechanism
for the "compensatory" increase in &globin synthesis is not
totally clear but may result from a "competition" among the
p-globin-like gene promoters for transcription factors.Iu
Exceptional& high HbA,. The mean level of HbA, in 879
carriers of P-thalassemia of diverse ethnic backgrounds was
5.08% f 0.39%.15' The highest observed value was 6.8%. In
184 black patients with P-thalassemia trait the mean HbA,
level was 4.97% 2 1.07%.15'
Some individuals with P-thalassemia trait have HbA,
concentrations that are significantlyhigher than these mean
levels. The exceptionally high HbA, levels are usually the
result of a unique and informative class of small deletions of
DNA that usually begin within the P-globin gene and
extend 5', removing the gene promoters. A summary of the
mutations so far described that are associated with high
HbA, are shown in Fig 3. These deletions may have direct
repeats, partial homologies, purine-rich regions, AT-rich
sequence, topoisomerase I1 recognition sites, and homologies to donor splice sequences in proximity to their 5' and 3'
ends. These have been postulated to lead to nonhomologous recombinations by several mechanism^."^ In contrast
to the very high H b 4 present with these 5' deletions, the
600-bp deletion in the 3' portion of the P-globin gene,
found in Asian Indians with Po-thalassemia, is associated
with typical HbA, levels.154A 3.4-kb deletion has its 5'
terminus between nucleotides -810 and -128 while the 3'
breakpoint is located between the Avu I1 and Xmn I sites
that lay 3' to the P-gene. This deletion removes both the
P-gene promoters and the 3' enhancer. The HbA, level in
the single heterozygote examined was 6.7%. Perhaps the
loss of the 3' enhancer element modulates the increase of
HbA, expected from the removal of the proximal promot-
2171
Fig 3. Positions of those fbglobin gene deletions, relative to the
p-globin mRNA capping site, that have been associated with unusually high levels of Hbh. The -3,400-bp deletion does not yet have
precisely defined 5' or 3' termini, and the 5' region of uncertainty is
indicated by the wavy line. This deletion appears to be characterized
by an HbA, level that is intermediate between the typical
p-thalassemias and those caused by the 5' deletions that have
breakpoints within the p-gene. The references for each deletion are:
532bp.";
1,393 bp."; 290 bp."'; 3,400 bp."; 4,237 bp,1'2; 12,622
bp.'". Modified with permission."
e r ~ .A' ~newly
~ described deletion of 44 bp begins in codon
24 or between codons 24 and 25 and extends 26 or 27 bases
into IVS I.153As the P-gene promoter is left intact, one
would predict that the level of HbAz would be similar to
that seen in the bulk of P-thalassemias. Unfortunately,
there is no information regarding HbAz levels in heterozygotes for this deletion.
The removal of the P-globin gene promoter sequences by
the 5' deletion may increase the likelihood that transcription factors, such as GATA-1,lS6bind the remaining 6- and
y-gene promoters, enhancing the transcription of these
genes. Alternatively, deletion-induced disruption of higherorder DNA or chromatin structure may make y- and
&promoters more accessible to the locus control region
(LCR). The LCR, a series of DNA'ase hypersensitive sites
that lie about 30 kb 5' to the €-globin gene, plays a major
role in governing transcription of the @-likegenes. The
LCR appears to interact with the P-like gene promoters in a
competitive f a ~ h i o although
n ~ ~ ~other
~ ~ mechanisms
~ ~ ~ ~ ~may
be po~sib1e.I~~
The absence of a functional P-promoter
might permit the LCR to interact with the &gene in cis,
enhancing its expression. These proposed explanations for
increased &gene expression in these interesting P-thalassemias due to 5' deletions are not mutually exclusive. Point
mutations in the P-globin gene promoter may be associated
with higher HbA, values than similar mutations elsewhere
in the P-gene. Perhaps, in a fashion analogous to the
situation where the P-gene promoters are deleted, these
mutations alter the binding of transcription factors and
favor expression of the &globin gene. In deletion HPFH
and SP-thalassemia, DNA rearrangements that reposition
the 3I-P-gene enhancer that binds GATA-1, in closer
proximity to the y-globin loci, have been postulated to
influence y-gene e x p r e s s i ~ n . ' ~The
~ . ' ~5' P-gene deletions
must have minor, if any, effect on the actions of this
enhancer.
This class of P-globin gene deletions also have the
potential to confound the prenatal diagnosis of sickle
hemoglobinopathies as they remove DNA that would hybridize with most allele-specific probes for the Psgene. Patients
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2172
STEINBERG AND ADAMS
heterozygous for both HbS and a 5' deletion Po-thalassemia
decrease in HbA, and an increase in mean corpuscular
volume (MCV).168,'69
The HbA, levels in untreated, nonanewould then appear to be homozygous for the psgene.
The HbA, levels in heterozygotes for these 5' P-gene
mic, hypothyroidism, cluster toward the low end of nordeletion thalassemias have been reported to range from
mal.169In hyperthyroidism, the combination of high HbA,
about 7% to 12%. We are unaware of the description of
and low MCV can be confused with P-thalassemia.
instances of bona fide HbA, levels that exceed the top of
Megaloblastic anemias have been associated with HbA,
concentrations that exceed normal level^.^"^^^^ The most
this range. Laboratory reports of HbA, values more than
15% or 20% are apt to be spurious or represent instances of
severely anemic patients have the highest HbA, values."'
HbC or HbE heterozygosity complicated by a-thalassemia.
However, the incidence of this finding seems low, the
Another theoretical possibility for exceptionally high level
magnitude of the increase above normal is slight, and, in
one study, the means of the HbA, levels in the normal and
of HbA, is the presence of a Miyada-type Hb, or PG-fusion
gene, where the point of crossing over leads to a globin
megaloblastic anemia groups were ~imi1ar.I~'Perhaps the
high HbA, of megaloblastic anemia is a result of more Hb
chain structurally identical ot
When this possibilsynthesis occurring in less mature erythroid precursor^.^'
ity was directly evaluated by restriction endonuclease mapping, it was not d 0 c ~ m e n t e d . lRecent
~~
follow-up of this
The HbA, levels appear to be increased in some instances of unstable H ~ s . ' ~ This
' , ' ~ is~ likely to be a postcase, after the reports of the effects of 5' P-gene deletions
on HbA,, and using more sensitive methods of analysis,
translational event where the unstable P-chain has difficulty
showed the presence of the 1.4-kb 5' P-gene deletion.162
forming @-dimers.
Malaria infestation and elevated HbA, levels have been
Miscellaneous causes of high HbA,. Some methods of
linked in some report^"^-'^^ but not other^.'^^,'^^ The best
measurement have reported elevated levels of HbA, in
controlled study casts doubt on such an ass~ciation.'~~
The
sickle cell trait (HbAS) and sickle cell anemia (HbSS)?6
association of hereditary spherocytosis and very high HbA,
Patients with H b S S - a - t h a l a ~ s e m i a ' ~ ~and
. ' ~ ~HbS-POlevels has also not been proven.'78
t h a l a s ~ e m i a ~ have
~ ~ ' ~ 'high HbA, concentrations. In the
former group of patients, the &chain competes more
CONCLUSION
effectively than the ps-chain for the limited quantities of
Genetic abnormalities that affect solely the &globin gene
a-chain. Mos~,~',"but not all,'66,167
methods of measuring
have no clinical significance for their carriers. But, tracing
HbA, in HbAS give results that are identical to or nearly
the origin and evolution of this locus has contributed to our
indistinguishable from normal.
knowledge of the plasticity that is inherent within the
HbA, levels appear to be consistently elevated in hyperthyroidism, albeit not as markedly as in P - t h a l a s ~ e m i a . ' ~ ~P-globin-like
. ~ ~ ~ ~ ~ ~ gene cluster. The variation in the level of
HbA, that accompanies the thalassemia syndromes and
Both the percentage and absolute amount of HbA, are
certain acquired diseases often provides very useful diagnosincreased, suggesting increased synthesis of the &globin
tic information. Thus, the vestigial 6-locus has achieved
chain, and an effect of thyroid hormone on &gene transcripclinical relevance that far eclipses its physiologic influence.
tion."' Euthyroidism, after treatment, is accompanied by a
-
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1991 78: 2165-2177
Hemoglobin A2: origin, evolution, and aftermath
MH Steinberg and JG 3d Adams
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