Thalassemia Alleles Research Articles

First report of a nonsense mutation at codon 15(TGG→TAG) in exon 1 of the β globin gene in a β thalassemia trait in State of Orissa, India

Prevention of β thalassemia requires knowledge of the molecular spectrum occurring in the population at risk. This knowledge is particularly necessary when prevention control is applied to a multiethnic population. For this purpose, we are analyzing different populations of Orissa (India). During the study we encountered a β thalassemia major patient (a child) who was doubly heterozygous for IVS I-5(G→C) and codon 15(G→A) alleles where codon 15(G→A) was for the first time found in the father of the patient. Also, the patient showed severe clinical abnormalities because of the severe nature of these β thalassemia alleles. This will provide further insights into β globin gene regulation and the genotype–phenotype relationship.

Co-inheritance of α-and β-thalassemia in Khuzestan Province, Iran

Background and aims: β-thalassemia is one of the most frequent hemoglobinopathies and single gene disorders in Iran. About 13 β globin mutations encompass 70–90% of mutations spectrum in Iran, the rest are rare or unknown. People who do not produce enough alpha globin protein chains have α-thalassemia. This is commonly found in Africa, the Middle East, India, Southeast Asia, southern China, and occasionally the Mediterranean region. There are normally four α globin genes, two on each chromosome 16. Individuals who have one or two abnormal alpha globin genes have α-thalassemia trait. The aim of this study was to detect α-thalassemia in β-thalassemia carriers during prenatal screening.Materials and methods: A total of 158 couples were diagnosed to be discordant α- and β-thalassemia carriers. We used the routine screening for thalassemia which includes full blood counts and indices, hemoglobin electrophoresis and measurement of Hb A2 level. The standard diagnostic marker for β-thalassemia is elevation of the Hb A2 level (>3·5%). Low mean corpuscular volume (MCV) and mean cell hemoglobin (MCH) with a normal Hb A2 indicate an α-thalassemia carrier. Staining for HbH inclusion bodies is also carried out as part of the screening for α thalassemia. The 59 and 39 ends of the breakpoint regions of the −α4·2 allele and the normal homologous segments were sequenced in selected individuals.Results: Of the 158 β-thalassemia partners, seven (4·4%) were found to have co-inheritance of α+-thalassemia, and three (1·9%) found to have co-inheritance of α0-thalassemia. Two pregnancies affected with Hb Bart's hydrops fetalis were terminated in the 158 couples. A sequence haplotype was found in all of the five Iranian −α4·2 thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the −α4·2 allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the α globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system. The major genotypes (−α4·2/αα, −α4·2/--MED, −α3·7/αα, −α3·7/−α3·7, αα/--MED and αα/αα) could be distinguished through the characteristic chromatograms generated by the WAVE system.Discussion: The results showed that molecular analysis must be used for accurate diagnosis of double heterozygotes in couples presumed to be discordant for α- and β-thalassemia on hematologic testing. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterized by Southern blotting or Gap-PCR.

THE Hb S/β+-Thalassemia Phenotype Demonstrates that the IVS-I (−2) (A>C) Mutation is a Mild β-Thalassemia Allele

We report a family in which two siblings are compound heterozygotes for Hb S [β6(A3)Glu→Val] and a rare β-globin mutation [IVS-I (−2) (A>C)]. Both patients had significant levels of Hb A, indicating that the IVS-I (−2) mutation is a relatively mild β+-thalassemia (β+-thal) allele. This mutation, in compound heterozygosity with Hb S, does not necessarily lead to a mild clinical course.

Haematological and clinical features of β‐thalassaemia associated with Hb Dhofar

Hb Dhofar is a variant haemoglobin (beta(29 (GGC-GGT) gly-gly), beta(58 (CCT-CGT) pro-arg)) associated with a thalassaemic phenotype and unique to the Sultanate of Oman. We report clinical and haematological data on 54 subjects with Hb Dhofar (37 heterozygotes, 14 homozygotes and three compound heterozygotes with a different beta-thalassaemia mutation). In heterozygotes, the level of Hb Dhofar ranged from 8.8% to 21.5%. All heterozygotes had Hb A2 > 3.5%, consistent with beta-thalassaemia trait. Hb Dhofar in homozygotes and compound heterozygotes ranged from 26% to 59.7%, with a peripheral film consistent with homozygous beta-thalassaemia. Age at presentation in homozygotes ranged between 6 months and 8 yr, with a majority presenting before 5 yr of age. All had splenomegaly and six (43%) had undergone splenectomy. All had some degree of frontal bossing and in particular, two patients with infrequent transfusions had marked thalassaemic facies and stunting of growth. Hb Dhofar can be mistaken for Hb D as the electrophoretic mobility is similar, but differs from it by a variable and reduced quantity of variant Hb in both heterozygotes and homozygotes. Clinical and haematological data suggest that this mutation behaves like a moderately severe beta(+) thalassaemia allele resulting in a thalassaemia intermedia phenotype.

Thalassemia intermedia associated with the Hb Constant Spring EE Bart's disease in pregnancy: A molecular and hematological analysis

We defined the molecular basis and analyzed hematological phenotype associated with an unusual form of thalassemia intermedia caused by interaction of the hemoglobin Constant Spring (Hb CS), homozygous Hb E and α°-thalassemia found in two unrelated pregnant Thai women. Both patients had moderate anemia and characteristic of thalassemia intermedia. Hb-HPLC analysis demonstrated in both cases, Hb E and Hb Constant Spring with 3–4% Hb Bart's. Hb F was marginally elevated (3–5%). Both of them were diagnosed hematologically as the Hb CS EE Bart's disease. DNA analysis revealed the homozygosity for Hb E in both cases and identified the Hb CS mutation in trans to the α°-thalassemia allele with the SEA deletion in one case and with the Thai deletion in another. The appearance of Hb-HPLC peak resembling the Hb CS in peripheral blood of the two cases indicated the ability to form a tetrameric Hb molecule between α CS and β E chains leading to a hybrid Hb namely the Hb E-CS (α 2 CSβ 2 E) with similar characteristics to Hb CS (α 2 CSβ 2 A). Hematological data of the patients were presented comparatively with other forms of related disorders in our series including 2 Hb H/Hb EE diseases, 16 homozygous Hb CS with and without Hb E, 14 Hb H diseases and 35 Hb H-CS diseases. Different genotype–phenotype correlations observed in these Thai patients with these disorders are illustrated.

Consanguinity affects selection of alpha-thalassemia genotypes and the size of populations under selection pressure from malaria

Background: In populations with α+-thalassemia gene deletion, the practice of consanguineous marriages is common.Aim: The study explored the impact of consanguinity (inbreeding) on the selection of α+-thalassemia genotypes in a computer model.Method: In a population under selection pressure from malaria, a single protective mutation (–α/αα genotype) was introduced among normal genotypes (αα/αα), and mating allowed to proceed. Heterozygote (–α/αα) and homozygote (–α/–α) children were 1.5 and 2.5 times more likely to survive malaria than those with normal genotypes. Using different coefficients of inbreeding (F, range 0–0.12), we examined the effect of population size, and the mean number of generations required for the homozygote frequency to reach 0.5.Results: On average, consanguineous populations were larger than randomly mating populations and the size was directly proportional to F. In more inbred populations,–α/–α homozygotes reached a frequency of 0.5 faster than in less inbred populations. As the frequency of the α+-thalassemia allele in a population increases, however, the positive effect of inbreeding on the population growth decreases.Conclusion: Under selection pressure from malaria, consanguinity may increase the speed of selection of–α/–α homozygotes and provide an advantage regarding population growth over non-consanguineous populations.

Prevalence of β-thalassaemia in subcastes of Indian Sindhis: Results from a two-phase survey

To estimate the prevalence of beta-thalassaemia in different subcastes of the Indian Sindhi population who, in general, have a high prevalence of this disease. A two-phase, community-based survey. Asymptomatic, Sindhi volunteers from Nagpur, central India, were recruited into the present study over a 7-year period. The first phase included the use of the Naked Eye Single Tube Red cell Osmotic Fragility Test (NESTROFT). Those positive for NESTROFT or those volunteering for haemoglobin A(2) (HbA(2)) quantification entered the second phase of the survey. Appropriate statistical methods for estimating prevalence from two-phase surveys were used. The prevalence of beta-thalassaemia carriers across the five major Sindhi subcastes varied substantially in the study population. Larkana Sindhis had the highest (17%) whereas Dadu Sindhis had the lowest (8%) frequency of the beta-thalassaemia allele. As a corollary, the projected incidence of beta-thalassaemia major in newborn babies greatly varied by the subcastes of the parents. Ethnic subgroups within populations known to commonly carry the beta-thalassaemia gene provide further information that is useful from epidemiological and public health perspectives.

A Novel beta-delta Globin Gene Fusion Leads to Over-Expression of Delta Globin Chain and a Mild Thalassemia Intermedia Phenotype When Co-Inherited with β0-Thalassemia.

Anti-Lepore hemoglobins are rare βδ fusion variants that arise from nonhomologous crossover during meiosis, resulting in a δ-βδ-β configuration in the β globin cluster on the short arm of chromosome 11. No such variant has previously been reported in Chinese. We studied 2 unrelated Chinese families with anti-Lepore due to a novel βδ fusion. A moderate increase of Hb A2 to between 16.8 and 19.2% of total Hb was seen in 4 clinically and hematologically normal carriers, while in 1 patient with concurrent β thalassemia allele and mild thalassemia intermedia phenotype, the Hb A2 was markedly raised to 42.2%. Potential βδ fusion gene was amplified using PCR with primer 5′ to β globin gene Cap site and 3′ to δ globin gene poly A region in these 5 subjects. Direct sequencing of the PCR products revealed a crossover between the β and δ globin gene within a 54 bp region spanning the junction of CAP and exon 1 in all 5 subjects. Active transcription of this novel βδ fusion gene was studied in 2 subjects (1 normal carrier and the patient with thalassemia intermedia). Using primer pair 5′ to β globin gene Cap site and at the 3′ end of δ globin gene exon 3, a 655 bp PCR product was obtained from cDNA in both subjects. Direct sequencing confirmed the presence of a βδ fusion mRNA. The predicted protein product of this fusion gene was identical to δ globin, since only the 5′ untranslated region was β specific. Determination of α/β mRNA ratios by quantitative real-time PCR was performed in 2 normal carriers (ratio 3.28 and 3.95) and the patient with thalassemia intermedia (ratio 25.59). These were compared with ratios ± standard deviation from 20 normal controls (1.92±0.55), 5 unrelated patients with β thalassaemia minor (17.23±5.38) and 3 unrelated patients with β thalassemia major (139.21±39.50). Mild down-regulation of the β gene in cis due to this interposed βδ fusion gene was demonstrated. Although heterozygotes of this novel anti-Lepore Hb had normal red cell indices and were clinically silent, compound heterozygotes with β0 mutation in trans produced a mild thalassemia intermedia phenotype with a markedly raised Hb A2 level that might mimic clinically mild Hb E-β+ thalassemia. Awareness of this new anti-Lepore Hb will help to resolve diagnostic problems in regions with significant prevalence of globin disorders.

Experience in preimplantation genetic diagnosis for exclusion of homozygous α° thalassemia

To report our experience in preimplantation genetic diagnosis (PGD) for the exclusion of homozygous alpha degrees thalassemia. PGD was performed on nine couples with alpha degrees thalassemia genotype undergoing assisted reproduction. Oocytes were aspirated after ovarian stimulation and fertilized by intracytoplasmic sperm injection. One or two blastomeres were biopsied from the six- to eight-cell embryo. Single cell multiplex PCR of the normal and alpha degrees thalassemia alleles was performed for first round, followed by semi-nested PCR of the respective alleles using 5'-end labelled fluorescent primers. Only those embryos with a blastomere diagnosed as having at least one normal allele were selected for transfer. One hundred and twenty-six blastomeres from 82 embryos were analyzed. The rates of allele dropout was 10.2% and PCR failure 12.7%. Fifty-eight embryos (70.7%) had at least one normal allele, of which 31 were transferred to 13 prepared cycles and one triplet pregnancy achieved. The triplets showed no ultrasound features of homozygous alpha degrees thalassemia at 18 weeks and were delivered in healthy condition by caesarean section at 34 weeks. Their genotypes were confirmed by cord blood analysis. PGD for alpha degrees thalassemia is possible by single cell PCR. The transfer and successful implantation of unaffected embryos ensure birth of disease-free babies.

Generation of a Novel Mouse Model of Cooley's Anemia.

A novel mouse model of Cooley's Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.

Prevalence of HFE mutations among the Thai population and correlation with iron loading in haemoglobin E disorder.

Co-inheritance of HFE mutations has a substantial role in iron overload in beta-thalassaemia carriers in north European populations where two HFE mutations, C282Y and H63D, are prevalent. In Thailand, there was little information about the allele frequency of HFE mutations. It is of interest to determine whether such determinants represent a potential risk in developing iron overload as nearly 40% of the Thai population carry either one of thalassaemia or haemoglobinpathy alleles. A total of 380 normal controls from five different regions including Bangkok were screened for the HFE C282Y, H63D and IVS5+1 G-->A alleles. In addition, 70 individuals with homozygous haemoglobin E (Hb EE) were also tested and their genotypes were correlated with levels of serum ferritin. H63D is the major HFE mutation found in the Thai population with an average allele frequency of 3% (range 1-5%). One individual was heterozygous for the splice site mutation IVS5 + 1 G --> A, and the C282Y allele was not detected. In the Hb EE group, five individuals had iron deficiency (ferritin <12 microg/L) and the remaining 65 individuals had a wide range of serum ferritin levels of 16-700 microg/L. Four individuals with Hb EE were heterozygous for the H63D allele. No significant difference in serum ferritin level was detected in this group with or without the HFE mutation (137.2 +/- 78 vs. 116.3 +/- 128 microg/L). HFE mutations are relatively uncommon among the Thai population, and the average allele frequency of the ancient H63D mutation is similar to that of other countries in this region. Because of their paucity, it appears that these alleles are less likely to be responsible for high ferritin levels and iron loading in individuals with Hb E related disorders.

Rapid, accurate genotyping of the common −α4.2 thalassaemia deletion based on the use of denaturing HPLC

Aims: To develop an alternative assay for specific genotyping of the −α4.2 thalassaemia deletion based on the DNA sequence features surrounding the breakpoint. Methods: The 5′ and 3′ ends of...

Genetic insights into the clinical diversity of beta thalassaemia.

Genetic insights into the clinical diversity of beta thalassaemia.

A New Hb Evanston Allele [α14(A12)Trp → Arg] Found Solely, and in the Presence of Common α‐Thalassemia Deletions, in Three Independent Asian Cases

Hb Evanston [α14(A12)Trp → Arg] is considered to be a rare α chain mutant, and was originally observed in two Black families in 1982, inducing a mild Hb H disease phenotype in a homozygous state for the − α3.7 deletion ( − αEvanston/ − α). The mutant, evidently linked with one of the two − α3.7 thalassemia (thal) alleles, was considered to be unstable and rapidly proteolyzed. We describe Hb Evanston in three new independent Asian cases, all induced by a TGG → CGG transition. In all cases the mutation is linked to the α1‐globin gene, either on a wild type allele or in linkage with the common − α3.7 and − α4.2 deletion alleles. The β/α ratio was balanced in the presence of the mutation only, and accordingly unbalanced in co‐inheritance with the deletion defects. Although a second independent mutation event on a − α3.7 or a − α4.2 deletion allele could not be excluded, we conclude that at least one independent Hb Evanston mutation has occurred on a wild type allele in the Asian populations. Unstable Hb tetramers tend to degrade and disappear during purification. Both Hb Evanston tetramers, formed in combination with normal β and δ chains, remain perfectly stable after extensive purification and concentration steps, suggesting an early posttranslational thalassemic effect, probably at the dimer/tetramer affinity level.

MOLECULAR CHARACTERIZATION OF Hb D-PUNJAB [β121(GH4)Glu→Gln] IN THAILAND

We describe the hematological and DNA characterization of Hb D-Punjab [β121(GH4)Glu→Gln] in Thailand. Nine patients from five unrelated families were studied; four patients were simple carriers of Hb D-Punjab, two were compound heterozygotes for Hb D-Punjab/β+-thalassemia;another two patients were double heterozygotes for Hb D-Punjab and α-thalassemia-2, and one patient was a compound heterozygote for Hb D-Punjab and Hb E [β26(B8)Glu→Lys]. Typical thalassemic indices with hypochromic microcytosis were observed in compound Hb D-Punjab/β+-thalassemia and Hb D-Punjab/Hb E but normal hematological profiles were observed in the remaining cases. DNA sequencing of the β-globin gene identified the G AA→C AA substitution at codon 121 causing Hb D-Punjab in all cases, and the −28 (A→G) mutation for the β+-thalassemia alleles. β-Globin gene haplotype analysis demonstrated, for the first time, that all these Asian βD-Punjab globin genes were associated with haplotype [−++−+++], previously undescribed in other populations. The finding of Hb D-Punjab in Thailand is compatible either with an independent origin of this abnormal hemoglobin or a spread of the Hb D-Punjab gene with a single origin among Asians.

A single tube multiplex PCR method to detect the common alpha + thalassemia alleles

To the editor: A single tube multiplex PCR method to detect the common + thalassemia alleles Alpha thalassemias are the commonest single gene disorders in humans. Two different deletions (−α3.7 and −α4.2) in the α globin genes (−α) on the short arm of chromosome 161 are the most

Spectrum of beta thalassemia mutations and their linkage to beta-globin gene haplotypes in the Indo-Mauritians.

The beta thalassemia alleles in 53 thalassemic Indo-Mauritian patients and their families consisting of 23 homozygous beta-thalassemia, 9 HbE/beta-thalassemia, 18 HbS/beta-thalassemia, 1 HbD/beta-thalassemia, 1 deltabeta/beta-thalassemia and 1 HbH/beta-thalassemia from the island of Mauritius were studied. Characterization by polymerase chain reaction-based reverse dot blot hybridization technique revealed that the IVS1-5 (G-->C) mutation accounted for 74% of the beta thalassemic alleles, while six other mutations occurred at much lower frequencies: HbE codon 26 (G-->A); 10.4%, codon 8/9 (+G); 3.5%, codon 30 (AGG-->ACG) also called IVSI (-1).G-->C; 3.5%, codon 15 (G-->A); 3.5%, codon 41/42 (-CTTT); 2.4% and -28 (A-->G); 2.4%. Association of these mutations to specific beta globin gene sequence framework and haplotype allowed to trace their ancestral link. These data are useful in future molecular screening of the population in view of implementing a thalassemia prevention and control program in Mauritius.

Comparative In Vivo Expression of β+-Thalassemia Alleles

Double heterozygotes who inherit one abnormal though stable β-globin variant in association with a molecularly identified β+-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular β+-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the β+-thalassaemia allele expressed as the MCH-βA in Pg βA-globin/β+-thalassemia allele/RBC = 0.5 MCH × Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the β+-87 (C→G) = 3.8 pg βA-globin/β+-thalassemia allele/RBC (n = 1); the β+IVS-I-1 (G→A) = 0.2 pg βA-globin/β+-thalassemia allele/RBC (n = 1); the β+ IVS-I-6 (T→C) = 2.9 pg βA-globin/β+-thalassemia allele/RBC (n = 7); the β+ IVS-I-110 (G→A) = 1.1 pg βA-globin/β+-thalassemia allele/RBC (n = 13), and the β+IVS-II-745 (C→G) = 1.74 pg βA-globin/β+-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other β+-thalassemia alleles from the literature. It can be seen that the MCH-βA value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in β-globin genes on pathophysiologically meaningful β-globin gene expression.

Is Hemoglobin Instability Important in the Interaction Between Hemoglobin E and β Thalassemia?

Hemoglobin E (HbE; α2β226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous β thalassemia. Yet, when it is inherited together with a β thalassemia allele, the resulting condition, HbE/β thalassemia, is sometimes characterized by a severe, transfusion-dependent thalassemia major. The severity of this interaction has not been explained. We have explored the possibility that it may reflect the instability of HbE consequent upon globin chain imbalance imposed by the β thalassemia allele. Time-course and pulse-chase globin chain synthesis studies at 37°C on peripheral blood and bone marrow suggest that hemoglobin instability is not significant in steady-state HbE/β thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/β+ thalassemia red blood cells age. Globin binding to membranes was assessed and only α globin chains were found, in contrast to other unstable hemoglobins in which both α and β chains were present. However, in experiments performed on blood from HbE/β thalassemics in the temperature range 39°C to 41°C, there was evidence of instability of HbE, a finding that was also observed in homozygous HbE. These findings suggest that the phenotype of HbE/β thalassemia is primarily the result of the interaction of two β thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia.© 1998 by The American Society of Hematology.

Is Hemoglobin Instability Important in the Interaction Between Hemoglobin E and β Thalassemia?

Hemoglobin E (HbE; 2β226glu-lys), globally the commonest hemoglobin variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous β thalassemia. Yet, when it is inherited together with a β thalassemia allele, the resulting condition, HbE/β thalassemia, is sometimes characterized by a severe, transfusion-dependent thalassemia major. The severity of this interaction has not been explained. We have explored the possibility that it may reflect the instability of HbE consequent upon globin chain imbalance imposed by the β thalassemia allele. Time-course and pulse-chase globin chain synthesis studies at 37°C on peripheral blood and bone marrow suggest that hemoglobin instability is not significant in steady-state HbE/β thalassemia; this is confirmed by density-gradient centrifugation studies that show no decrease in HbE levels relative to HbA as HbE/β+ thalassemia red blood cells age. Globin binding to membranes was assessed and only  globin chains were found, in contrast to other unstable hemoglobins in which both  and β chains were present. However, in experiments performed on blood from HbE/β thalassemics in the temperature range 39°C to 41°C, there was evidence of instability of HbE, a finding that was also observed in homozygous HbE. These findings suggest that the phenotype of HbE/β thalassemia is primarily the result of the interaction of two β thalassemia alleles; however, hemoglobin instability may be important during febrile episodes, contributing to worsening anemia. © 1998 by The American Society of Hematology.