Download - BLOOD The Journal of - bioquest.org fileBLOOD The Journal of The American SocieQ of Hematology VOL 78, NO 9 NOVEMBER 1, 1991 REVIEW ARTICLE Hemoglobin A,: Origin, Evolution, and Aftermath

BLOOD The Journal of The American SocieQ of Hematology

VOL 78, NO 9 NOVEMBER 1, 1991

REVIEW ARTICLE

Hemoglobin A,: Origin, Evolution, and Aftermath

By Martin H. Steinberg and Jun ius G. Adams 111

H E MELANGE of hemoglobins present in the erythro- T cytes of humans includes hemoglobin A, (HbA,),’., a tetramer of a- and &globin chains (a26,). Its unique characteristics reside in the n~n-a-chain.~ HbA, is physio- logically 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

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 @-globin gene 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).

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 present (Mybp).32 The next duplication gave rise to the divergence of the a-globin genes and occurred approximately 450 Mybp.32 The P-globin gene is thought to have duplicated about 180 to 200 Mybp into an ancestral gene for E- and y-globin and an ancestral gene for 6- and P-gl~bin.’~ Approximately 110 to 130 Mybp, the E/? parent gene diverged to establish the E and y lineages.

Evolution 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.38 42

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.M4n It 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

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 IVSII ) .~~

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.44 However, Petes” has made an interesting observation concerning the structural variants of HbA,.

genes).” 44 4‘47 cu

Gene conversion and the &globin gene.

From the Department of Medicine, University of Mississippi School

Submitted June 17, 1991; acceptedAugust 21, 1991. Supported by Research Funds of the Department of Veterans Affairs. Address reprint requests to Martin H. Steinbe%, MD, (151), VA

This is a US government work. There are no restrictions on its use.

of Medicine, and VA Medical Center, Jackson, MS.

Medical Center, 1500 E Woodrow Wilson Dr, Jackson, MS 39216.

0006-4971 191 17809-0049$0.00/0

Blood, Vol78, No 9 (November 1). 1991: pp 2165-2177 2165

2166 STEINBERG AND ADAMS

Chromosome 11

H 5kb

Fig 1. The arrangement of the plike globin gene cluster.

Two of the then-described 10 &-globin variants 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 1 of 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. An- other 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.s2 This 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.’’

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 reticulo- cyte~.’~.’~ These experiments were extended by fractionating bone marrow erythroblasts into fractions of different levels

Synthesis of the &globin chain.

P I My b.p.

A -190

A A -130

p c* 85 (Mammalianradiation) e Y 6 Fig 2. The evolutionary history of the &-globin gene.

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-a- globin 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 6- and 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.61 This 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

HEMOGLOBIN A, 2167

adult Hbs within the mammalian @-globin gene cluster inactivates the y-globin genes as 6- and P-gene transcription is activated.62

HbA, has functional properties that are nearly identical to those of HbA. It has similar oxygen affinity, Bohr effect, and ~ooperativity.6~,~~ Its response to 2,3 bisphosphoglycerate is also similar to that of HbA.65 The thermal stability is greater than that of HbA.& HbA, inhibits polymerization of HbS.67 The residues 622 (Ala) and 687 (Gln) appear to be the important inhibitory sites. In instances where the HbA, is exceptionally high, and in the presence of elevated HbF levels, the combination of these two Hbs may modulate the phenotype of HbS-Po-thalassemia.68 The positive charge of HbA, may endow it with properties similar to other positively charged Hbs, such as HbC, relative to its interaction with the erythrocyte HbA, has a higher affinity for erythrocyte membrane band 3 than does HbA.70 While the interaction of HbC with the membrane is believed to determine the pathophysiologic properties of HbAC and HbCC cells,71 the concentrations of HbA, make it doubtful that it can meaningfully affect cation transport and mean corpuscular Hb concentration.

The Lepore Hbs are the products of SP hybrid globin genes and have provided insight into the mechanisms of decreased &globin synthesis. In 1958, Gerald and Diamond7* showed that the individuals who carried this variant exhibited hematologic manifestations identical to P-thalassemia, but had an elec- trophoretically slow-moving Hb that comprised 10% to 15% of the total hemolysate. Subsequently, B a g l i ~ n i ~ ~ showed that the non-cu-globin chain of Hb Lepore had &globin sequences at its amino terminus and P-globin sequences at its carboxyl terminus. The Lepore Hbs most likely arose from a nonhomologous crossing over event in which the P-globin gene from one chromosome mispaired with the &globin gene of the other chromosome during synapsis. The result of this recombination is the deletion of a segment of DNA approximately 7 kb in length from a point within the transcribed portion of the &globin gene to a corresponding point in the P-globin gene. Therefore, the Lepore chromosome does not have a normal 6- or @-globin gene.

Current concepts of how the Lepore SP fusion gene variants produce the phenotype of thalassemia are that the &globin gene sequences located at the 5’ portion of these fusion genes result in their characteristically reduced rate of ~ynthesis.’~ The major difference in this region, as mentioned previously, is that in the &globin gene, the CCAAT box promoter sequence is CCAAC. Another puzzling feature of the Lepore globin chain was that is was synthesized at a higher rate than the &globin chain of HbA,. This increased synthesis of the hybrid globin is most likely due to the presence of the second intervening sequence of the P-globin gene.@ The rare homozygous forms of Hb Lepore, as well as combination of Hb Jipore with other P-thalassemia genes, are usually expressed clinically as moder- ately severe forms of thalassemia major.74

The reciprocal product of the crossover that produces the

Function of HbA, and the &globin chain.

Hybrids of the p and &globingenes.

Lepore globin genes produces a chromosome with a 8-PS-P configuration. Two of these “anti-Lepore” variants have been found, Hb Miyada” and Hb P N i l o t i ~ . ~ ~ . ~ ~ Because these individuals have a normal P-globin gene, they do not exhibit a thalassemic phenotype. The anti-Lepore globin gene has the P-globin gene promoter, so synthesis of these globins would be expected to be near that of the P-globin gene. In Hb P Nilotic heterozygotes, the variant Hb was found to comprise 21% to 28% of the hem~lysate.~~.~’ This proportion closely approximates that expected if the P Nilotic globin chain was synthesized at nearly the same rate as the normal P-globin chain. It has been shown that the synthesis of Hb P Nilotic decreases in reticulocytes as compared with bone marrow cells,77 but this decrease in synthesis is not a major determinant of the total synthesis of this globin because it appears to accumulate in the expected amounts in red blood cell (RBC) precursors. The synthesis of the PG-globin chain of Hb Miyada is apparently synthesized somewhat less efficiently than Hb P Nilotic, compris- ing only 17% of the hemolysate in heterozygous individu- a l ~ . ~ ~ The synthesis of this abnormal globin was also found to be greatly decreased in reticulocytes when compared with bone marrow cells.78

The non-a-globin chain of Hb Parchman is also informative in this regard. If Hb Parchman arose a double crossover as initially suggested, it should contain the &globin gene promoter, IVS-I from the p-globin gene, and IVS-I1 from the &globin gene. The non-cu-globin chain of Hb Parchman is synthesized at the same rate as the normal &globin chain, supporting the suggestion of Kosche et aI@’ that the decreased synthesis of the &globin chain is due to the presence of the CCAAC promoter and the &globin gene IVS-11.

The known structural variants of the &globin gene are depicted in Table 1. Although these variants may be unstable or have elevated oxygen affinity, they are not associated with a clinically significant phenotype because, as mentioned previously, HbAz has no detectable effect on the oxygen transport of the RBC due to its low proportion. Because HbA, variants have no effect on phenotype, the number of structural variants of the &globin gene is far less than that of the a- or P-globin gene.

&Globin variants.

CLINICAL FEATURES OF HBA,

The &chain of HbA, contains two additional positive charges compared with the P-chain of HbA. This facilitates its separation by electrophoretic and chromatographic methods that rely on charge differences to resolve proteins from one another. Hb electrophoresis on starch or polyacrylamide gels or cellulose acetate membranes affords wide separation of HbA, from HbA and HbF and a means to quantify accurately this minor Hb ~omponent .~~. ’~ Acid agar gel electrophoresis does not resolve HbA, from HbA. The ease of working with cellulose acetate membranes, their commercial availability and reasonable cost, makes this the current electrophoretic method of The low level of HbA, in erythrocytes, and the narrow range separating normal from abnormal, causes methods of measurement that are facile from the

Measurement of HbA,.

STEINBERG AND ADAMS 2168

Table 1. Structural Variants of the &Globin Gene

Amino Acid Mutation Residue No. Abnormality Hb Name Population Frequency % Variant Reference

GIG + CCG 1 (NA1) Val + Ala A, Niigata Japanese XR - 184 185

A A I + nqc! or AAG 12 (As) Asn + Lys A, NYU Eastern European R 1 .o 186 - GGC + CGC 16(A13) Gly+Arg A; Black American q = .009 1 .o 6.7.10 CIG + GAG 20 (82) Val -+ Glu A2 Roosevelt Iraqi XR 1 .o 187 GCA -+ G F 22 (84) Ala + Glu A, Flatbush Black American R - 188

GGT -+ GAT 24 (B6) Gly + Asp A, Victoria Iraqi XR 1.3 189 GGT + GAT 25 (87) Gly + Asp A, Yokoshima Japanese XR 44% of HbA, 190

CAT -+ CGT 2 (NA2) His + Arg A, Sphakia Cretan Canadian XR - Indian q = .004

Babinga pygmies q = ,024

- GAG + -@G 43 (CD2) Glu + Lys A, Melbourne Italian XR 1.2 191 GAT + G F 47 (CD6) Asp + Val A, Parkville ? XR - 192 CCT -+ CGT 51 (D2) Pro + Arg A, Adria Italian XR 1.6 193 - GGT + CGT 69 (E13) Gly + Arg A, Indonesia Malayan q = .001 1.4 194,195

GAG -+ GIG 90 (F6) Glu -+ Val A, Honai Japanese XR 0.8 196 - GTG -+ ATG 98 (FG5) Val + Met A, Wrens’ Black American XR 0.2 197

Sumatran q = .024

- GAT + &IT 99 (Gl) Asp + Asn A, Canadat East Indian XR 1.8 198 CGC + CAC or CATS 116 (G18) Arg -+ His A, Coburg Italian XR 1.4 199

G&I + GIA 121 (GH4) Glu -+Val A, ManzanaresI Spanish XR 0.4 200 - CAA + GAA 125 (H3) Gln + Glu A, Zagreb Yugoslavian XR 1.1 20 1 GGT + GAT 136 (H14) Gly -+ Asp A, Babinga Babinga pygmies q = ,0007 1 .o 202

- CGC + JGC 116 (G18) Arg + Cys A, Corfu Greek R 0.5-1.5 204

GCT + GAT 142 (H20) Ala + Asp A, Fitzroy Greek XR 1.4 203

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.84 Refrigeration and freez- ing may reduce the HbAz percentage in stored hemoly- sates.” 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 immunologi~a l ly .~~~~’~~ This technique 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.

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 synchro- nously 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

HbA, in health and disease.

HEMOGLOBIN A, 2169

components of HbA, are present and can be quantified by HPLC and isoelectric foc~s ing ."~~"~ As with HbA,,, these glycohemoglobins are elevated in poorly controlled diabet- ics.

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.

Hb tetramer assembly follows rapidly on the formation of dimers consist- ing 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

Low HbA,.

Posttranslational causes of reduced HbA,.

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

Acquired a-Thalassemia

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 reple- tion. 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.Izs This, and other hematologic differences between these groups, sug- gests 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.

a-Thalas- semia 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

Genetic conditions associated with low HbA,


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 &thalassemia- causing 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 &-thalassemia may cause the HbA, to be normal in the presence of P-thalassemia.I3l Interactions between a-thalassemia and p-thaIassemia may 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

7.2-kb deletion, extending from 3’ 6p through IVS-2 of the 8-gene.”.lm

~ ~ 1 3 7 . 1 3 1 1 1

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 HbAz level 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

HEMOGLOBIN A, 2171

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 a8 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

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 significantly higher 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.154 A 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-

Exceptional& high HbA,.

Fig 3. Positions of those fbglobin gene deletions, relative to the p-globin mRNA capping site, that have been associated with unusu- ally 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.153 As 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,lS6 bind the remaining 6- and y-gene promoters, enhancing the transcription of these genes. Alternatively, deletion-induced disruption of higher- order 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 @-like genes. The LCR appears to interact with the P-like gene promoters in a competitive f a ~ h i o n ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ although 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 expres 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 hybrid- ize with most allele-specific probes for the Ps gene. Patients


heterozygous for both HbS and a 5' deletion Po-thalassemia would then appear to be homozygous for the ps gene.

The HbA, levels in heterozygotes for these 5' P-gene deletion thalassemias have been reported to range from about 7% to 12%. We are unaware of the description of instances of bona fide HbA, levels that exceed the top of this range. Laboratory reports of HbA, values more than 15% or 20% are apt to be spurious or represent instances of HbC or HbE heterozygosity complicated by a-thalassemia. Another theoretical possibility for exceptionally high level of HbA, is the presence of a Miyada-type Hb, or PG-fusion gene, where the point of crossing over leads to a globin chain structurally identical ot When this possibility was directly evaluated by restriction endonuclease mapping, it was not d0c~mented . l~~ Recent follow-up of this case, after the reports of the effects of 5' P-gene deletions on HbA,, and using more sensitive methods of analysis, showed the presence of the - 1.4-kb 5' P-gene deletion.162

Some methods of measurement have reported elevated levels of HbA, in sickle cell trait (HbAS) and sickle cell anemia (HbSS)?6 Patients with HbSS-a-thala~semia'~~. '~~ and HbS-PO- tha l a s~emia~~~ '~ ' have high HbA, concentrations. In the former group of patients, the &chain competes more effectively than the ps-chain for the limited quantities of a-chain. Mos~,~'," but not all,'66,167 methods of measuring HbA, in HbAS give results that are identical to or nearly indistinguishable from normal.

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 . ' ~ ~ . ~ ~ ~ ~ ~ ~ Both the percentage and absolute amount of HbA, are increased, suggesting increased synthesis of the &globin chain, and an effect of thyroid hormone on &gene transcription."' Euthyroidism, after treatment, is accompanied by a

Miscellaneous causes of high HbA,.

decrease in HbA, and an increase in mean corpuscular volume (MCV).168,'69 The HbA, levels in untreated, nonane- mic, hypothyroidism, cluster toward the low end of normal.169 In hyperthyroidism, the combination of high HbA, and low MCV can be confused with P-thalassemia.

Megaloblastic anemias have been associated with HbA, concentrations that exceed normal level^.^"^^^^ The most severely anemic patients have the highest HbA, values."' However, the incidence of this finding seems low, the magnitude of the increase above normal is slight, and, in one study, the means of the HbA, levels in the normal and megaloblastic anemia groups were ~imi1ar.I~' Perhaps the high HbA, of megaloblastic anemia is a result of more Hb synthesis occurring in less mature erythroid precursor^.^'

The HbA, levels appear to be increased in some instances of unstable H ~ s . ' ~ ' , ' ~ ~ This is likely to be a posttranslational event where the unstable P-chain has difficulty forming @-dimers.

Malaria infestation and elevated HbA, levels have been linked in some report^"^-'^^ but not other^.'^^,'^^ The best controlled study casts doubt on such an ass~ciation. '~~ The association of hereditary spherocytosis and very high HbA, levels has also not been proven.'78

CONCLUSION

Genetic abnormalities that affect solely the &globin gene have no clinical significance for their carriers. But, tracing the origin and evolution of this locus has contributed to our knowledge of the plasticity that is inherent within the P-globin-like gene cluster. The variation in the level of HbA, that accompanies the thalassemia syndromes and certain acquired diseases often provides very useful diagnostic information. Thus, the vestigial 6-locus has achieved clinical relevance that far eclipses its physiologic influence.

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201. Juricic D, Crepinko I, Efremov GD, Lam H, Webber BB, Headlee MG, Huisman THJ: Hb A,-Zagreb or a&, 125 (H3) Gln +Glu, a new 6 chain variant in association with SP- thalassemia. Hemoglobin 7:443, 1983

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Download - BLOOD The Journal of - bioquest.org fileBLOOD The Journal of The American SocieQ of Hematology VOL 78, NO 9 NOVEMBER 1, 1991 REVIEW ARTICLE Hemoglobin A,: Origin, Evolution, and Aftermath

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