The Hemoglobin E Thalassemias Suthat Fucharoen 1 and David J. Weatherall 2 1 Thalassemia Research Centre, Institute of Science and Technology for Research and Development, Mahidol University, Bangkok, Thailand 2 Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom Correspondence: [email protected]Hemoglobin E (HbE) is an extremely common structural hemoglobin variant that occurs at high frequencies throughout many Asian countries. It is a b-hemoglobin variant, which is produced at a slightly reduced rate and hence has the phenotype of a mild form of b thalassemia. Its interactions with different forms of a thalassemia result in a wide variety of clinical disorders, whereas its coinheritance with b thalassemia, a condition called hemo- globin E b thalassemia, is by far the most common severe form of b thalassemia in Asia and, globally, comprises approximately 50% of the clinically severe b-thalassemia disorders. A s discussed by Williams and Weatherall (2012), HbE occurs at an extremely high frequency in many countries in Asia. Because there is also a high frequency of different b- thalassemia alleles in these populations, the co- inheritance of HbE and b thalassemia, HbE b thalassemia, occurs very frequently. Similarly, because different forms of a thalassemia are also very common in these countries, HbE also occurs together with them, producing a com- plex series of phenotypes. The first description of HbE b thalassemia appeared in a paper by Minnich and her col- leagues in 1954 under what, at the time, was the rather surprising title “Mediterranean Anaemia: A study of 32 cases in Thailand” (Minnich et al. 1954). In the same year, the first electrophoretic identificationofHbEwasreportedindependently (Itano et al. 1954). The first detailed clinical de- scription of HbE b thalassemia was reported in 1956 by Chernoff and colleagues (1956). Much later, groups in Thailand began a detailed analysis of the interaction of the various forms of a thal- assemiawith HbE, which result in a complex series of phenotypes, most of which are much milder than HbE b thalassemia (Wasi et al. 1969). MOLECULAR PATHOLOGY AND PROPERTIES OF HEMOGLOBIN E At least in vitro, HbE appears to be mildly un- stable and shows increased sensitivity to oxi- dants (Frischer and Bowman 1975). However, in vitro studies of hemoglobin synthesis do not show evidence of instability similar to that found in other unstable hemoglobin variants, although HbE is unstable at increased tempera- tures, similar to those that would occur in a wide range of infective diseases (Rees et al. 1998). The whole-blood oxygen dissociation curves of Editors: David Weatherall, Alan N. Schechter, and David G. Nathan Additional Perspectives on Hemoglobin and Its Diseases available at www.perspectivesinmedicine.org Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011734 Cite this article as Cold Spring Harb Perspect Med 2012;2:a011734 1 www.perspectivesinmedicine.org on June 13, 2020 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/ Downloaded from
Transcript
The Hemoglobin E Thalassemias
Suthat Fucharoen1 and David J. Weatherall2
1Thalassemia Research Centre, Institute of Science and Technology for Research and Development, MahidolUniversity, Bangkok, Thailand
2Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS,United Kingdom
Hemoglobin E (HbE) is an extremely common structural hemoglobin variant that occurs athigh frequencies throughout many Asian countries. It is a b-hemoglobin variant, which isproduced at a slightly reduced rate and hence has the phenotype of a mild form of b
thalassemia. Its interactions with different forms of a thalassemia result in a wide variety ofclinical disorders, whereas its coinheritance with b thalassemia, a condition called hemo-globin E b thalassemia, is by far the most common severe form of b thalassemia in Asia and,globally, comprises approximately 50% of the clinically severe b-thalassemia disorders.
As discussed by Williams and Weatherall(2012), HbE occurs at an extremely high
frequency in many countries in Asia. Becausethere is also a high frequency of different b-thalassemia alleles in these populations, the co-inheritance of HbE and b thalassemia, HbE b
thalassemia, occurs very frequently. Similarly,because different forms of a thalassemia arealso very common in these countries, HbE alsooccurs together with them, producing a com-plex series of phenotypes.
The first description of HbE b thalassemiaappeared in a paper by Minnich and her col-leagues in 1954 under what, at the time, was therather surprising title “Mediterranean Anaemia:A study of 32 cases in Thailand” (Minnich et al.1954). In the same year, the first electrophoreticidentificationofHbEwasreportedindependently(Itano et al. 1954). The first detailed clinical de-scription of HbE b thalassemia was reported in
1956 by Chernoff and colleagues (1956). Muchlater, groups in Thailand began a detailed analysisof the interaction of the various forms of a thal-assemiawithHbE,whichresult inacomplexseriesof phenotypes, most of which are much milderthan HbE b thalassemia (Wasi et al. 1969).
MOLECULAR PATHOLOGY ANDPROPERTIES OF HEMOGLOBIN E
At least in vitro, HbE appears to be mildly un-stable and shows increased sensitivity to oxi-dants (Frischer and Bowman 1975). However,in vitro studies of hemoglobin synthesis do notshow evidence of instability similar to thatfound in other unstable hemoglobin variants,although HbE is unstable at increased tempera-tures, similar to those that would occur in awiderange of infective diseases (Rees et al. 1998). Thewhole-blood oxygen dissociation curves of
Editors: David Weatherall, Alan N. Schechter, and David G. Nathan
Additional Perspectives on Hemoglobin and Its Diseases available at www.perspectivesinmedicine.org
Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011734
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homozygotes for HbE appear to be normal orvery slightly right-shifted. HbE is synthesized ata slightly reduced rate and homozygotes showmild globin-chain imbalance, similar to that ob-served in b-thalassemia heterozygotes. It iscaused by a base substitution at codon 26 ofthe b-globin gene, GAG-AAG, which results inthe substitution of lysine for glutamic acid. Thismutation also activates a cryptic splice site thatcauses abnormal messenger RNA processing(Orkin et al. 1982). Because the normal donorsite has to compete with this new site, the level ofnormally spliced bE messenger RNA is reduced(Traeger et al. 1980), resulting in the clinicalphenotype of a mild form of b thalassemia.
The heterozygous state for HbE is character-ized by minimal morphological abnormalitiesof the red cells and normal red cell indices;HbE constitutes 25%–30% of the hemoglobin(Fig. 1). Homozygotes for HbE have hypochro-mic microcytic red cells with significant mor-phological abnormalities including increasednumbers of target cells (Fig. 2). They are mildlyanemic and the overall hematological findingsare very similar to those of heterozygous b thal-assemia.
THE INTERACTIONS OF HEMOGLOBIN EWITH DIFFERENT FORMS OF THALASSEMIA
Although HbE alone does not cause any signifi-cant clinical problems, its interactions with var-ious forms of a and b thalassemia produce avery wide range of clinical syndromes of varying
severity. The molecular basis for the differentforms of a and b thalassemia, which are coin-herited with HbE, are described by Thein (2012)and Higgs (2012).
The various interactions of HbE and a thal-assemia, which have been defined in the Thaipopulation, and occur elsewhere in SE Asia, aredescribed by their particular hemoglobin con-stitutions (Table 1). Heterozygotes for HbE,which are also heterozygous for aþ thalassemia(2a/aa), have similar levels of HbE to HbEheterozygotes, whereas those that are heterozy-gous fora0 thalassemia (2/aa) have mild thal-assemic red cell changes and the level of HbEranges between 19% and 21%. HbE heterozy-gotes who also inherit the genotype of HbHdisease (2/2a) have a marked decrease ofHbE in the 13%–15% range and a thalassemicdisorder of intermediate severity, which is calledHbAE Bart’s disease.
Hemoglobin E homozygotes who coinheritthe heterozygous state for aþ or a0 thalassemiahave a mild hypochromic microcytic anemiawithslightly elevated levels of HbF. Those who coin-herit the genotype of HbH disease have a thal-assemic disorder of intermediate severity withmoderate anemia and elevated levels of HbF andBart’s, a condition called EF Bart’s disease.
The compound heterozygous state for HbEand b thalassemia, HbE b thalassemia, is a re-markably heterogenous disease with a pheno-type ranging from mild anemia to the most se-vere forms of b-thalassemia major (Weatherall
Figure 1. The peripheral blood film in hemoglobin Etrait showing normal red cell morphology.
Figure 2. The peripheral blood film in the homozy-gous state for hemoglobin E showing large numbersof target cells.
S. Fucharoen and D.J. Weatherall
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and Clegg 2001). This condition may also be co-inherited with a variety of different forms of athalassemia. The coinheritance of the heterozy-gous states for aþ and a0 thalassemia have anameliorating effect on the disease, whereas thosewho also inherit the genotype of HbH diseasehave a form of HbEF Bart’s disease, as describedabove.
These complex interactions, which are sum-marized in Table 1, produce three significantclinical disorders; HbAE Bart’s disease, HbEFBart’s disease, and HbE b thalassemia.
HbAE BART’S DISEASE
This thalassemia syndrome is characterized bythe presence of HbA, HbE, and Hb Bart’s, andresults from the interaction of the genotype ofHbH disease (see Higgs 2012; Vichinsky 2012)with heterozygous HbE (Wasi et al. 1967; Thon-glairuam et al. 1989). Two common subtypes ofHbAE Bart’s disease have been observed: aþ
thalassemia/a0 thalassemia—A/E and a0 thal-assemia/Hb Constant Spring—A/E. Clinically,HbAE Bart’s disease is similar to HbH disease,and like the latter, the a0 thalassemia/Hb Con-stant Spring interaction is more severe (see Vi-chinsky 2012). However, in this syndrome thereare no hemolytic crises during stress similar tothose seen in HbH disease. The amount of HbEis decreased to 13%–15%. This is because a-globin chains have a lower affinity for bE thanbA-globin chains. Small amounts of Hb Bart’sare always present in this genotype and intra-erythrocytic inclusion bodies (HbH inclusions)can be demonstrated in �5% of the erythro-cytes, indicating the presence of small amountsof HbH that is insufficient to be detected byelectrophoresis.
The diagnosis of this disorder requires de-tailed family studies together with DNA analysisto define the type ofa thalassemia. Managementis similar to HbH disease (see Vichinsky 2012).
HbEF BART’S DISEASE
HbEF Bart’s disease is characterized by the pres-ence of HbE, HbF, and Hb Bart’s (Fucharoenet al. 1988b). HbE constitutes �80% and HbF
10% of the hemoglobin; the remainder is HbBart’s. The presence of Hb Bart’s indicates thatthere are excess g-globin chains. However, noinclusion bodies or HbH are present, probablybecause the abnormal bE-globin chains do notform tetramers. Four genotypes of HbEF Bart’sdisease have been identified. They result frominteractions between the genotype for HbH dis-ease, either a0 thalassemia/aþ thalassemia ora0 thalassemia/Hb Constant Spring, with ei-ther homozygous HbE or HbE b thalassemia.Hb Constant Spring and small amounts of HbAmay be observed in patients with the a0 thalas-semia/Hb Constant Spring and HbE bþ thalas-semia genotype. To differentiate among thesegenotypes, family studies and further investiga-tion by DNA analysis are required.
Overall, the interactions of the different ge-notypes for HbH disease with the homozygousstate for HbE produce relatively mild forms ofthalassemia intermedia, not dissimilar to HbHdisease (see Vichinsky 2012). Their interactionswith HbE b thalassemia are more complex andclinically variable, and are discussed in moredetail in the next section.
HEMOGLOBIN E b THALASSEMIA
In general, HbE b thalassemia is a thalassemiasyndrome of intermediate severity with avery het-erogeneous clinical spectrum. Two types havebeen described, depending on the presence or ab-sence of HbA. In HbE, b0 thalassemia, bA-globinchains are not synthesized and the condition ischaracterized by the production of HbE andHbF without detectable HbA; HbE constitutesbetween 30% and 70% of the hemoglobin withthe remainder HbF. Variable amounts of HbA aredetected, in addition to HbE and HbF, in HbEbþ
thalassemia. Different bþ thalassemia mutationsresult in variable severity of the disease, reflectingdifferent levels of HbA.
Pathophysiology
The pathophysiology of HbE b thalassemia re-flects both the reduced output of HbE togetherwith the added globin-chain imbalance conse-quent on the coinheritance of b thalassemia.
S. Fucharoen and D.J. Weatherall
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Although early studies suggested that HbE isslightly unstable and may precipitate underconditions of oxidative stress, later biosyntheticanalyses showed little evidence of its instabilityin the red cells of patients with HbE b thalasse-mia. The one exception was if the cells were ex-posed to increased temperatures at the level thatmight be encountered in severe forms of infec-tion at which there was evidence of instability(Rees et al. 1998). However, the remarkableameliorating effect on the phenotype that re-sults from the coinheritance of a thalassemiaor other modifiers of the degree of globin chainimbalance (see later section) suggests that defec-tive b chain synthesis is the major factor in thepathophysiology of this condition (reviewed byWeatherall and Clegg 2001). Like other forms ofsevere b thalassemia, there is marked expansionof the erythroid bone marrow with ineffectiveerythropoiesis. Interestingly, using a combina-tion of electron-microscopic and immunocy-tochemical studies, Wickramasinghe and Lee(1997) demonstrated that the erythroblast in-clusions in the bone marrow of patients withthis condition consist entirely of precipitated a
chains; there is no evidence of coprecipitation ofbE chains. Therefore the mechanisms of damageto red cells and their precursors are similar tothose described in other forms of b thalassemia(see Nienhuis and Nathan 2012).
Recent studies have suggested that thereare important differences in the compensatorymechanisms between HbE b thalassemia andother forms of severe thalassemia intermedia.In particular, patients with HbE b thalassemiaappear to be able to compensate for anemia by aright shift in their oxygen dissociation curves,unlike those with many other forms of b-thal-assemia intermedia (Allen et al. 2010). Thisprobably reflects both the properties of HbEand the lower mean levels of HbF that occurin HbE b thalassemia compared with otherforms of b-thalassemia intermedia. Studies ofthe erythropoietin response to anemia in thiscondition have shown that hemoglobin leveland age are independent variables with respectto the erythropoietin level and that there is arelative reduction in response to a particularhemoglobin level with aging (O’Donnell et al.
2007). This may be at least partly responsible forsome of the remarkable phenotypic heterogene-ity observed during the early years of develop-ment in babies with HbE b thalassemia.
Clinical Features
One of the most striking features of HbE b thal-assemia is its remarkable clinical heterogeneity.At one end of the spectrum, there are patientswhose clinical course is almost indistinguishablefrom that of severe b-thalassemia major; where-as at the other end, there are patients who growand develop normally without the need forblood transfusion, albeit often at a relativelylow hemoglobin level.
At birth, infants with severe HbE b thalas-semia are asymptomatic because HbF levels arehigh. As HbF production decreases and is re-placed by HbE at 6–12 months of age, anemiawith splenomegaly develops. Signs of impairedgrowth appear during the first decade of life. Theinitial complaints vary from patient to patient,and several symptoms usually appear simulta-neously (Table 2). Most common are the de-velopment of a mass in the left upper quadrantand pallor. With time and without transfusions,anemia, jaundice, hepatosplenomegaly, growth
Table 2. Presenting symptoms in 378 HbE b-thalas-semia patients in Thailand
Symptoms Number %
1. Pallor 150 39.72. Fever 72 19.13. Abdominal mass 36 9.54. Abdominal pain 23 6.15. Combined: Abdominal mass
and pain21 5.6
6. Yellow eyes ( jaundice) 16 4.27. Edema 9 2.48. Pregnancy with anemia 3 0.89. Bone pain 3 0.8
retardation, and thalassemic facies evolve (Table3). Absence of secondary sexual development iscommon and chronic leg ulcers are sometimesobserved. These manifestations, which are sec-ondary to decreased oxygen delivery to tissue,ineffective erythropoiesis, and iron overload, re-semble those of b-thalassemia major.
Patients with the milder forms of HbE b
thalassemia tend to grow and develop reason-ably well during early childhood and are fullyactive. There may be some delay in the pubertalgrowth spurt and in the appearance of secondarysexual characteristics but they usually attain areasonable height and sexual maturation. Fur-ther work is required to determine whether theydevelop the later complications that have beendescribed in older patients with other formsof thalassemia intermedia, such as renal disease,pulmonary hypertension, cerebral infarcts, andothers (see Musallam et al. 2012). Certainlysome of them accumulate iron through in-creased absorption and may develop associatedendocrine complications including diabetes.Furthermore, there is undoubtedly phenotypicinstability in the early years of life; in a seriesof children observed over 15 years with HbE
b thalassemia in Sri Lanka, at least 20 caseschanged from a mild to a more severe phenotypeduring the first 15 years of life.
Laboratory Findings
The steady-state hemoglobin levels in HbE b
thalassemia range widely between the differentphenotypes, from 3 g/dl or less to as high as11 g/dl. The red-cell indices and morphologicalchanges are similar to those described in otherforms of severe b thalassemia (Fig. 3). Hemo-globin analysis reveals a preponderance of HbEwith low levels of HbA in those who have inher-ited a bþ-thalassemia mutation. The mean levelof HbF in 200 patients from Sri Lanka was ap-proximately 28%, ranging from less than 10% to50%; even higher levels of HbF have been re-ported from Thailand. Family studies revealthe carrier state for HbE in one parent and forb-thalassemia trait in the other. The laboratoryfindings associated with different complicationsare discussed in the following sections.
Complications
Hypersplenism
Splenomegaly, together with pooling of red cellsand their increased rate of destruction, is extreme-ly common. In the more severe phenotypes, itoften progresses rapidly from the first few years
Figure 3. The peripheral blood film in hemoglobin Eb thalassemia after splenectomy showing numerousnucleated red cells and a high platelet count.
Table 3. Clinical signs in 378 adult HbE b-thalasse-mia patients in Thailand
of life, whereas in the milder phenotypes, al-though the spleen is palpable, it usually doesnot attain a size greater than 5–6 cm below thecostal margin. Much less common, and usually inthe milder phenotypes, splenomegaly may slowlyincrease over 10–20 years and only become aproblem later in life. As well as resulting in in-creasing anemia, progressive enlargement of thespleen is quite often associated with pain anddiscomfort in the left upper quadrant.
Infection
Patients with HbE b thalassemia are susceptibletoviral, bacterial, and fungal infection, which arecommon causes of mortality (Aswapokee et al.1988a,b). In splenectomized patients, septi-cemia can be veryacute and overwhelming, lead-ing to death in a short period. Gram-negativeand gram-positive bacteria are frequent causesof septicemia.Fungal infectionwith Pythium canlead to arterial occlusion and gangrene of the legs(Sathapatayavongs et al. 1989; Wanachiwanawinetal.1993).The mechanismthatcauses increasedsusceptibility to infections is not known. Recentstudies have suggested that patients with HbE b
thalassemia may be more prone to infection byboth P. falciparum and P. vivax malaria, particu-larly the latter, and that those who have under-gone splenectomy may be even more susceptible(O’Donnell et al. 2009).
Cardiac Disease
Approximately half of the patients with HbE b
thalassemia in Thailand die of heart failure. Thisis associated with failure of other organs, delayedgrowth and sexual maturation, hepatomegaly,and endocrinopathies. Organ failure resultsfrom iron deposition in the heart and other tis-sues (Vannasaeng et al. 1981; Sonakul et al. 1988;Thakerngpol et al. 1988) resulting from increasedabsorption and blood transfusion. Myocardialiron deposition is usually limited, occurring pri-marilyas small granules in perinuclear areas, withlater accumulation throughout the muscle fibers,predominantly subepicardial and occasionallysubendocardial (Sonakul et al. 1984). The smallamount of iron deposited in the heart is in
marked contrast to enormous iron depositionin the liver and pancreas. Cardiomegaly is pro-portional to the severity of anemia and systolicmurmurs are frequently present (Jootar and Fu-charoen 1990). Chronic pericarditis followingupper respiratory tract infection is frequently en-countered, more commonly in splenectomizedpatients. A pericardial rub may be detected, oftentransiently. Intractable pericardial effusion mayfollow, causing cardiac tamponade and failure,and requiresaspiration. Inavery fewcases, chron-ic constrictive pericarditis develops, requiringsurgical intervention.
Heart rate variability (HRV) has been de-veloped to determine cardiac autonomic func-tion and is applied to investigate patients withthalassemia major (De Chiara et al. 2005). Stud-ies of the HRV in HbE b thalassemia have ob-tained similar results to those in thalassemiamajor (Rutjanaprom et al. 2009). The depressedHRV compared to normal suggests that HRVmay be a marker of cardiac sympatho-vagal im-balance as well as an early indicator of cardiacinvolvement in both thalassemia major andHbE b thalassemia.
Pulmonary hypertension and right heartfailure are discussed in the following sections.
Hypoxemia
The majority of splenectomized HbE b-thalas-semia patients in Thailand develop hypoxemiawith low arterial pO2 (Wasi et al. 1982). Plateletcounts in these patients are double those ofnonsplenectomized patients; young and largerplatelets are also observed in the absence ofthe spleen. Platelet microaggregates have beendetected in the circulation of these splenecto-mized patients. One hypothesis for the patho-genesis of hypoxemia in HbE b thalassemia isthat platelets increase in number, are youngerand more active after splenectomy, and aggre-gate in the circulation and in the pulmonaryvasculature. Substances released during plateletaggregation may cause constriction of the ter-minal bronchioles leading to decreased oxygen-ation and hypoxemia. Administration of aspirinto inhibit platelet aggregation reduces the de-gree of hypoxemia in the majority of cases
Hemoglobin E Thalassemias
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(Fucharoen et al. 1981), suggesting that aspirinshould be routinely given to splenectomizedpatients with HbE b thalassemia. Interestingly,this combination of pulmonary hypertensionand hypoxemia, together with right ventricularfailure, has not been observed so frequently inother populations, suggesting that other factorsmay be involved in the Thai population.
Thromboembolism
Autopsy findings in a large number of Thaipatients with HbE b thalassemia revealed strik-ing pulmonary artery occlusion (Fig. 4) (Sona-kul et al. 1980). Thromboembolism in HbE b
thalassemia seems to involve platelets, a reactivethalassemic red cell surface, coagulation fac-tors, and abnormal endothelium (Butthep et al.2002; Pattanapanyasat et al. 2004).
Hypertension, Convulsions, andCerebral Hemorrhage
Some patients in Thailand develop hyperten-sion, convulsions, and cerebral hemorrhage af-ter transfusion of 2 units or more of blood (Wasiet al. 1978). This complication may develop aslate as 2 weeks after multiple transfusions, sug-gesting that blood volume overload is not thecause of hypertension. Monitoring blood pres-sure during and after blood transfusions withprompt antihypertensive intervention has re-duced deaths from this complication. This syn-drome has not been reported in other popula-tions.
Extramedullary Hemopoiesis
Erythropoiesis is massively increased to 10–15times normal because anemia stimulates ery-thropoietin production. Extensive erythropoie-sis can be found in the liver, spleen, bone mar-row, and in extramedullary sites. Erythropoieticmasses in the spinal canal can cause spinal cordcompression and paraplegia, and when they oc-cur intracranially, convulsions may result (Issa-ragrisil et al. 1981). Massive erythropoiesis leadsto fragility and distortion of the bones and de-creases bone density because of osteoporosis andosteomalacia, as observed in irregularly trans-fused patients (Pootrakul et al. 1980).
Jaundice, Gallstones, and Cholecystitis
Some HbE b-thalassemia patients have severeand persistent jaundice in the absence of defin-able liver disease. It turns out that this is a resultof the homozygous inheritance of the TA(7) al-lele of the promoter of the glucuronyltransferase1 gene, a polymorphism that is particularly com-mon in Sri Lanka (Premawardhena et al. 2001).These patients have a highly significant increasein the incidence of gallstones. Homozygosity forthe TA(7) allele occurs in 10%–25% of somepopulations of Africa and the Indian subconti-nent but at a much lower frequency in SoutheastAsia (Premawardhena et al. 2003). Stones arefound in approximately 50% of HbE b-thalas-semia patients in Thailand (Chandcharoensin-Wilde et al. 1988). For the detection of biliarycalculi, ultrasonography is more sensitive thanoral cholecystography and plain abdominalfilms. Cholecystitis and ascending cholangitismay occur with abdominal pain, fever, and in-creasing jaundice (Vathanopas et al. 1988).
Iron Overload
Iron overload occurs commonly (Pootrakulet al. 1981). Excessive iron accumulates becauseof blood transfusions and enhanced gastrointes-tinal absorption. The skin is darkened and irondeposition occurs in the bone marrow, liver,spleen, heart, pancreas, and elsewhere (Sonakulet al. 1988; Thakerngpol et al. 1988). The relatedcardiac complications were discussed earlier.
Figure 4. Pulmonary vascular occlusion in hemoglo-bin E b thalassemia after splenectomy.
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Although liver fibrosis from iron overload iscommon, ascites and other signs of cirrhosisare very rare. Diabetes mellitus, secondary toiron deposition in the pancreas, frequently devel-ops in untreated adult patients (Vannasaeng et al.1981). A terminal wasting stage occurs in somepatients who survive to their fourth and fifthdecades. They develop severe skin pigmentation,poor appetite, weight loss, and increasing ane-mia, and eventually die. This is believed to resultfrom organ failure caused by uncontrolled tissueoxidation from chronic, severe iron overload.
Recently, it has been possible to obtain moredirect data about the liver iron concentration inHbE b thalassemia using spin density projec-tion assisted R2-MRI technology (Olivieri et al.2011). These studies showed a marked variationin hepatic iron levels even in patients who hadreceived only minimal transfusion. They alsounderlined the rather poor agreement betweenhepatic iron levels as measured in this way andserum ferritin values. Clearly, a great deal morework is required to try to determine the reasonsfor the variable rate of iron accumulation byincreased intestinal absorption and, in particu-lar, whether this reflects polymorphisms in oneor more of the genes that are involved in theregulation of iron absorption.
Other Endocrine Disorders
As well as diabetes there are other importantendocrine disorders that occur as a result ofiron loading. In particular hypothyroidism andhypoparathyroidism are quite common and theformer is frequently associated with growth re-tardation. It is vital, therefore, to carry out reg-ular assessments of thyroid and parathyroidfunction in patients with HbE b thalassemia,regardless of the severity of their phenotype.
Genotype–Phenotype Interaction
Definition of Severity
Despite seemingly identical genotypes, com-pound heterozygotes for b thalassemia andHbE have remarkably variable phenotypes. No-table are variations in the degree of anemia,growth, development, hepatosplenomegaly, and
transfusion requirements. A novel scoring sys-tem based on six independent parameters—he-moglobin level, age at disease presentation, ageat receiving first blood transfusion, requirementfor transfusion, spleen size, growth and develop-ment—was able to separate patients into threedistinctive severity categories: mild, moderate,and severe. The scoring system consisting ofsix clinical criteria scored as 0, 0.5, 1, or 2, ac-cording to clinical presentation. HbE b-thalas-semia patients with total scores ranging from 0to 3.5, 4 to 7, and 7.5 to 10 are grouped as mild,moderate, and severe cases, respectively. The se-vere patients are very anemic and are usuallytransfusion dependent; some may have markedgrowth retardation, whereas the mild cases havemild anemia and usually have normal growthand development (Sripichai et al. 2008).
As indicated by recent studies in Sri Lanka,the application of a clearly defined scoring sys-tem for severity combined with a long periodof observation and genetic and environmentalanalysis (Premawardhena et al. 2005; Olivieriet al. 2010) should help us to understand thefactors that determine the broad range of se-verity of HbE b thalassemia.
b-Thalassemia Mutations
Althoughb0 thalassemia is caused by many mu-tations, all result in absence of b-globin chainproduction by the abnormal gene. b0 thalas-semia is more severe than bþ thalassemia, inwhich a wide range of b-globin chain produc-tion is observed. In most countries with a highfrequency of HbE b thalassemia, the commonb-thalassemia mutations are either b0 thalas-semia or bþ thalassemia associated with verysmall amounts of b-globin chain synthesis.The fact that there is still considerable clinicalheterogeneity in these patients is clearly not aresult of variation in b-globin chain synthesisdirected by the chromosome containing the b-thalassemia mutation. There are, however, cer-tain milder forms of bþ thalassemia associatedwith much higher levels of b-chain productionand, when inherited together with HbE, pro-duce a much milder form of HbEb thalassemia.The phenotypes and hemoglobin findings in
Hemoglobin E Thalassemias
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patients of this type are summarized by Weath-erall and Clegg (2001).
Coinheritance of a Thalassemia
The concomitant inheritance of a thalassemiaor Hb Constant Spring may be responsible forless severe anemia and a milder phenotype inHbE b0 thalassemia (Winichagoon et al. 1985,1993). Coinheritance of a0 thalassemia withHbE b0 thalassemia may lead to so mild a con-dition that the individuals do not have a clinicalabnormality that requires medical attention.Similar findings have been observed in Sri Lan-kan populations, in which the coinheritance ofthe heterozygous state for aþ thalassemia hasbeen found to result in a remarkable degree ofamelioration of the clinical severity of HbE b
thalassemia (Premawardhena et al. 2005).
Association with Increased HbF
Coinheritance of determinants that increaseHbF expression can ameliorate the severity ofHbE b thalassemia. Inheritance of a chromo-some with the C ! T polymorphism that re-sults in an Xmn-1 cleavage site at position 2158to the Gg-globin gene is associated with increasedHbF and milder anemia (Winichagoon et al.1987). Two copies of this allele (Xmn-1 þ/þ)are necessary to produce a significant clinical ef-fect. Increased expression of the Gg-globin genewas also detected in the Xmn-1 þ/þ patients.This increase of g-globin gene activity reducesthe overall globin chain imbalance and thus ame-liorates the anemia. The association between theXmn-1 þ/þ genotype and a highly significantincrease in the absolute level of HbF and a milderphenotypehas also been observed inpatients withHbEb thalassemia in Sri Lanka (Premawardhenaet al. 2005). It is likely that several other polymor-phisms will have this effect (see later section andSankaran and Orkin 2012).
Amount of Alternatively SplicedbE-Globin mRNA
An underproduction of b-globin chains fromthe bE-globin gene strongly suggests that al-ternative RNA splicing is of functional signifi-
cance. The percentage of alternative spliced bE-globin mRNA was determined by the reversetranscriptase polymerase chain reaction tech-nique in 14 patients with the same thalassemiamutation (Winichagoon et al. 1995). Prelimi-nary results showed abnormally spliced bE-glo-bin mRNA in patients with severe symptomsand low hemoglobin levels between 2.9% and6.1%, whereas those with higher hemoglobinlevels had values from 1.6% to 2.6%. The ma-jority of patients with the Xmnl-negative geno-type had more severe anemia and a higherpercentage of abnormally spliced bE-globinmRNA. This indicated that the amount of alter-natively spliced E-globin mRNAwas a more im-portant factor in determining severity of anemiathan the pattern of Xmnl polymorphism or thelevel of HbF. Recently, Tubsuwan et al. used theallele-specific RT-qPCR to studybE-globin geneexpression and found that the correctly to aber-rantly spliced bE-globin mRNA ratio in 30% ofmild HbE b-thalassemia patients was higherthan that of the severe patients. It appears there-fore that the splicing process of bE-globin pre-mRNA differs among HbE b-thalassemia pa-tients and serves as one of the modifying factorsfor disease severity (Tubsuwan et al. 2011). Itwill be important to determine whether thisphenomenon occurs in other ethnic groups.
Pyrimidine 50 Nucleotidase Deficiency
In a Bangladeshi family, an individual homozy-gous for both HbE and pyrimidine 50 nucleo-tidase deficiency was found. The patient had asevere hemolytic anemia in contrast to HbE ho-mozygotes. Globin chain synthesis experimentsshowed that the mechanism underlying the in-teraction between these two genotypes was amarked decrease in the stability of HbE in py-rimidine 50 nucleotidase-deficient red bloodcells. In these cells, free a-globin chains butnot bE-globin chains accumulated on the mem-brane. It was hypothesized that the marked in-stability of HbE in the enzyme-deficient cellsresulted from oxidant damage to mildly unsta-ble HbE (Rees et al. 1996). Clearly this interac-tion also has the potential to modify the phe-notype of HbE b thalassemia.
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Recently, a genome-wide association study(GWAS) was performed in 618 Thai HbE b0-thalassemia patients using the Illumina Human610-Quad BeadChips array (Nuinoon et al.2010). DNAs were extracted from 383 severeand 235 mild phenotypes, by a validated scoringsystem, after the exclusion of a thalassemia.Twenty-three single nucleotide polymorphisms(SNPs) in three independent genes/regionswere identified as being significantly associatedwith the disease severity. The highest associationwas observed with SNPs in the b-globin genecluster (chr.11p15). The second was identifiedin the intergenic region between the HBS1L andMYB genes (chr.6q23), and the third region waslocated in the BCL11A gene (chr.2p16.1). Anassociation to HbF levels with SNPs in thesethree regions was observed. This result suggeststhat several genetic loci act in concert to influ-ence HbF levels of HbE b0-thalassemia patients(see Sankaran and Orkin 2012).
Variation in Adaptation to Anemia andEnvironmental Factors
As already mentioned, there appears to be areduction in erythropoietin production in rela-tionship to a similar hemoglobin level with ag-ing, a finding which may explain some of thephenotypic instability during the early years oflife. Whether there is a genetic component tothe magnitude of erythropoietin response re-mains to be determined. Undoubtedly, patientswith HbE b thalassemia adapt more readilyto low hemoglobin levels than those with otherforms of b-thalassemia intermedia (Allen et al.2010). Although as is the case for most inheritedhemoglobin disorders, the role of the environ-ment in modifying the phenotype has been ne-glected; recent studies suggest that patientswith HbE b thalassemia are more susceptibleto malaria infection, particularly that causedby P. vivax, than age-matched controls in thepopulation (O’Donnell et al. 2009). There isalso reasonable evidence that those who havebeen exposed to malaria tend to have largerspleens and fall into the more severe phenotypiccategories. Much more work is required to fur-
ther elucidate environmental factors that maymodify the phenotype.
Conclusion
The genotypic factors that can be used to predicta mild phenotype in HbEb thalassemia are mildbþ-thalassemia mutations, the coinheritance ofa thalassemia, the polymorphisms associatedwith HbF production such as homozygosityfor Xmn-l restriction site 50 to the Gg-globingene and the BCL11A gene. Some complicationsof the disease such as severe jaundice are alsoaffected by genetic modifiers. And it is also clearthat at least some factors that modify the re-sponse to anemia or the environment of pa-tients with this disease are also responsible forphenotypic diversity. But although progress hasbeen made, it is still only possible to explain partof its wide phenotypic diversity.
Treatment
Because HbE b thalassemia has such a variablephenotype and patients with this disorder—probably because they have relatively lower lev-els of HbF and reflecting the oxygen affinity ofHbE—are able to adapt to anemia better thanpatients with other forms of thalassemia inter-media, it is vital to observe babies and youngchildren with this condition after presentationfor a reasonable period before deciding on thebest approach to management. It is importantto remember that they may present with a par-ticularly low hemoglobin level consequent to arecent infection, and it is particularly importanttherefore not to establish them on a regulartransfusion until their steady-state hemoglobinlevel and level of growth and degree of spleno-megaly has been assessed. Particularly in areaswhere malaria is endemic it is also important toexclude chronic P. vivax infection as a possiblecause of rapidly progressive splenomegaly.
The hemoglobin level alone should notbe the major factor in initiating transfusion.Rather, the broader picture should be consid-ered with particular attention to growth failure,lack of activity, and the earlier appearance ofskeletal change. If it is clear that the patient
Hemoglobin E Thalassemias
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requires regular transfusion the regimen to befollowed, including chelation, is similar to thatfor the management of thalassemia major (seeBrittenham and Olivieri 2012). Those who donot require transfusion should be maintainedon folic acid supplements and advised aboutthe early treatment of infective episodes. Al-though some patients with increasing splen-omegaly and evidence of hypersplenism maybenefit from splenectomy, this should be avoid-ed where possible because of the particularlyhigh risk of infection.
Patients who do not require regular transfu-sion should have serum ferritin or hepatic MRIestimations at least twice per year. Increasediron levels should be controlled by intermittentcourses of chelating agents.
Hydroxyurea therapy may increase HbF lev-els (Fucharoen et al. 1996), although recentstudies in other populations have shown thatthis effect is not great, even when combinedwith erythropoietin. For those who present ear-ly with severe disease, bone marrow transplan-tation remains an important option (Issaragrisil1994; Leelahavarong et al. 2010).
Rapidly expanding extramedullary hemo-poietic masses, particularly involving the brainor spinal cord, require urgent treatment byblood transfusion, hydroxyurea, or possibly, ra-diotherapy. Limited experience in those withprofound jaundice as a result of genetic inabilityto conjugate bilirubin suggest that, at least insome cases, very low doses of phenobarbitonemay be helpful.
The first successful report of gene therapyin thalassemia involved a patient with HbEb thalassemia (Cavazzana-Calvo et al. 2010).Although the patient showed clinical improve-ment and did not need further blood transfu-sion, it is not yet clear whether this is mainlybecause of the action of the inserted “normal”b-globin gene. The improvement also seems tobe at least partly a result of an increase in HbFlevels by an unknown mechanism.
Prevention of HbE b Thalassemia
Effective prevention programs for thalassemiahave been demonstrated in many countries
in which carrier rates for different types ofthalassemia are very high (see Brittenham andOlivieri 2012). In Thailand, screening hasimproved by using an automatic high-perfor-mance liquid chromatography (HPLC) system(Fucharoen et al. 1998). In recent years, a na-tionwide program in Thailand has been devel-oped to prevent homozygous b thalassemia,HbE b thalassemia, and Hb Bart’s hydrops fe-talis, with encouraging results (Tongsong et al.2000). The prospective screening consisted ofosmotic fragility (OF) and HbE screening testsin pregnant women, followed by testing thehusbands of the women with a positive re-sult. Subsequently, the OF test was replaced bymean corpuscular volume (MCV) when auto-mated cell counters became available nation-wide. If both partners of the couple have a pos-itive result, further diagnostic tests by HPLCand genotyping of the carrier are carried out.A pregnancy in which both partners of thecouple are carriers is considered as a couple atrisk, and further detailed counseling and pre-natal diagnosis is offered for the severe thalas-semia syndromes (Fucharoen and Winichagoon2007).
Since the program began, the number ofnew cases of thalassemia in Thailand has grad-ually decreased. In the first few years of theprogram, its cost-effectiveness was evaluatedand it was found that among a total pregnantpopulation of 21,000 individuals that werescreened, 80 affected fetuses had been identi-fied and the pregnancy terminated. The totalcost of the prevention program was about U.S.$257,140, and the cost of management of theseaffected cases, if they had been born, would havebeen U.S. $7,200,000. The cost-benefit ratiowas 1:28, which indicates a highly cost-effectiveproject.
CONCLUSIONS
HbE b thalassemia is a major public healthproblem in Southeast Asia and in other Asiancountries. Although some progress has beenmade toward a better understanding of its path-ophysiology and clinical management a greatdeal remains to be learned. Recent work has
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made it absolutely clear that there must be othergenetic modifiers to be discovered that are re-sponsible for the variable phenotype. A betterapproach to predicting the phenotype is urgent-ly required, particularly if prenatal diagnosis isto be widely used for the control of this condi-tion and, even more so, if experimental forms ofgene therapy become available in the future.Because it may be some time before there aremore definitive forms of treatment, it is impor-tant to utilize the information that we alreadyhave more effectively. For example, in malariousareas it will be very important to conduct trialsof malaria prophylaxis. With particular respectto the phenotype of patients with this conditionearly in life, and because recent evidence sug-gests that the erythropoietin response to anemiatends to decline with age, the possibility of tran-sient periods of transfusion during maximumerythroid expansion should be seriously consid-ered. And because genetic evidence indicatesthat the phenotype in this condition may beimproved quite dramatically with only a modestincrease in steady-state hemoglobin level, moreefforts should be directed at trying to raise theHbF level in these patients.
Other Interactions of HbE
This article has considered the most commonand important clinical interactions of HbE. Be-cause it is so common it is not surprising thatmany rare interactions between this variant anda wide variety of structural hemoglobin vari-ants and related conditions have been reported.The resulting disorders are described brieflyin a recent review (Fucharoen and Weather-all 2009).
ACKNOWLEDGMENTS
This work is supported by the Office of theHigher Education Commission and MahidolUniversity under the National Research Univer-sity Initiative and the National Center for Ge-netic Engineering, Thailand and The WellcomeTrust, UK. We thank Liz Rose for her help inpreparing this work.
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Hemoglobin E Thalassemias
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