A novel molecular basis for β thalassemia intermedia poses new questions about its pathophysiology

Among the complex genetic interactions that underlie the intermediate forms of β thalassemia there have been several reports of individuals heterozygous for β thalassemia who are either homozygous for triplicated^1-7  or heterozygous for quadruplicated^6,8-10  α globin gene arrangements. However, because the frequency of these extended α gene arrangements is probably low in most populations, genotypes of this kind are thought to be only rare causes of these forms of β thalassemia.

In a survey of the thalassemia population of Sri Lanka it was found that 2% to 3% are carriers for triplicated or quadruplicated α globin genes.¹¹  As part of a detailed analysis of the genotypes of patients with β thalassemia attending the National Thalassaemia Centre, Kurunegala, Sri Lanka, 2 patients were encountered who were heterozygotes for a β thalassemia mutation. Further analysis revealed that in one case there was a previously undescribed genotype, that is, homozygosity for quadruplicated α globin genes combined with heterozygosity for β thalassemia. The other patient was homozygous for a triplicated α globin gene arrangement and heterozygous for β thalassemia.

Here, we report clinical, hematologic, and molecular analyses of these 2 patients and compare the findings with other forms of β thalassemia intermedia in Sri Lanka, notably patients with triplicated α globin genes who also had hemoglobin (Hb) E β thalassemia and a group of 29 patients with Hb E β thalassemia with 4 α globin genes. The findings raise some important questions about the mechanisms that underlie the complex clinical phenotypes of the intermediate β thalassemias, particularly the level of fetal hemoglobin.

Family H was referred to the Thalassaemia Centre for further assessment of a boy with an intermediate form of β thalassemia. Family G was ascertained during an analysis of the molecular forms of β thalassemia in transfusion-dependent patients in Kurunegala; the propositus was found to have only a single β globin gene mutation and hence a full family study was carried out. The patients with Hb E β thalassemia were ascertained through a detailed analysis of all the patients with severe thalassemia attending the center.

Approximately 3- to 10-mL blood samples were collected into EDTA (ethylenediaminetetraacetic acid). Hematologic analysis was carried out using an automated counter (microdiff 8; Coulter, Miami, FL), and the remainder of the blood was frozen at -20°C and transported on dry ice to Oxford, United Kingdom, for further analysis.

Hemoglobin analysis was carried out using cation-exchange high-performance liquid chromatography (HPLC; BioRad Variant, BioRad, Hercules, CA). α/β+γ globin chain synthesis was measured by standard methods¹¹ ; red cells were incubated with ³H leucine in Sri Lanka, washed, frozen, and transported to Oxford on dry ice for globin-chain separation. DNA was extracted, and the β thalassemia mutations were characterized by the amplification refractory mutation system (ARMS) or DNA sequencing, as previously described.¹²  α Globin gene analysis, assessment of the -158 C→T (Xmn-1) polymorphism upstream from the ^Gγ globin gene, and promoter polymorphisms of the uridine diphosphate (UDP) glucuronosyltransferase 1A1 (UGT1A1) promoter were analyzed by previously described methods.^11-13  The ratio of α to β globin mRNA was assessed by an RNase protection assay.¹⁴  Serum erythropoietin (Epo) levels were measured using a Quantine Diagnostics enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) (normal range, 1-14 mIU/mL).

These studies were approved by the Central Oxford Research Ethics Committee, the Oxford Tropical Research Ethics Committee, the Ethics Committee of the Faculty of Medicine, University of Colombo, and the Sri Lankan Ministry of Health. Informed consent was provided according to the Declaration of Helsinki.

The results of hematologic, hemoglobin synthesis, and mRNA studies in the 2 families are summarized in Table 1.

The propositus (II-1; Table 1), a 4½-year-old boy and the product of a consanguineous marriage, first presented at the age of 4 months, when his parents noticed pallor and jaundice. A clinical diagnosis of thalassemia was made without further investigation, and he was given a single-unit transfusion. In the absence of any more symptoms the parents did not seek further medical assistance for several years. When seen at the age of 5 years, he was asymptomatic with good exercise tolerance. On examination he was found to be at the 75th centile for height with a slight delay in bone age, 3 years at a chronologic age of 4 year 8 months. His spleen and liver were palpable at 5 cm and 2 cm below the left and right costal margins, respectively. His hemoglobin level has ranged between 55.0 and 78.0 g/L (5.5 and 7.8 g/dL), and his blood picture showed marked hypochromia, microcytosis, nucleated red cells, and extremely abnormal red cell morphology. Hemoglobin analysis revealed Hb A₂ and Hb F levels of 4.3% and 4.6%, respectively. The Epo level was 130 mIU/mL at an Hb level of 65.0 g/L (6.5 g/dL).

DNA analysis indicated that the propositus is heterozygous for the β thalassemia mutation, IVS1-5 (G→C). α Globin gene analysis (Figure 1) showed that he is homozygous for a quadruplicated arrangement. The α/β globin synthesis ratio was 3.2:1 and the mRNA ratio was 18.1:1. He was also found to have the genotype -/- at the ^Gγ Xmn-1 site.

A family study (Table 1) was restricted to the parents; there were no siblings. Neither parent showed any physical abnormalities, and, in particular, the spleen was not palpable. However, both were anemic, the mother, with a hemoglobin value of 76.0 g/L (7.6 g/dL), was more severely affected than her husband. The blood films of both parents showed marked hypochromia and microcytosis with poikilocytosis, both had elevated Hb A₂ levels, and DNA analysis revealed that they were heterozygotes for the same β globin gene mutation as their son and for the quadruplicated α globin gene arrangement. Subsequent iron studies, carried out in an attempt to explain the greater degree of anemia in the mother, showed that she had a serum iron value of 17.1 μmol/L (95.7 μg/dL), within the normal range.

The propositus (II-1; Table 1), a 16-year-old boy, presented at the age of 5 years with a febrile episode. There was no history of exercise intolerance, and his growth and development appeared to have been satisfactory. However, the detection of pallor and splenomegaly led to a clinical diagnosis of thalassemia and, without further investigation, he was commenced on regular monthly blood transfusions. On examination, aged 16 years, he was short but showed no obvious thalassemic bone changes. He was noted to be markedly icteric. His height and weight were below the third centile, and his bone age was delayed to 11 years 6 months at a chronologic age of 16 years. He had no secondary sexual development, and his spleen and liver were palpable 5 cm and 2 cm below the left and right costal margins, respectively. Hematologic studies, carried out at least 6 months after stopping transfusion, showed a hemoglobin value of 65.0 g/L (6.5 g/dL); marked hypochromia, microcytosis, and poikilocytosis with nucleated red cells on the blood film; an elevated level of Hb A₂ and an Hb F of 2.0%. The Epo level was 143 mIU/mL at a Hb level of 62.0 g/L (6.2 g/dL).

A liver biopsy showed a hepatic iron concentration of 24.07 mg/g dry liver, and there was extensive iron-loading and fibrosis (3/6, Ishak staging). Transfusions were stopped, and a course of deferoxamine was instituted at a dose of 35 mg/kilo/24 hours. Off transfusion he has functioned extremely well at a hemoglobin value of 60 to 70 g/L (6-7 g/dL) and is continuing chelation therapy. It appears, therefore, that his delayed growth and sexual development reflect endocrine damage secondary to severe iron loading resulting from transfusion without adequate iron chelation.

Analysis of his DNA showed that he is heterozygous for a β thalassemia mutation, codon 15 (-T), homozygous for a triplicated α globin gene arrangement (Figure 1), ^Gγ Xmn-1^-/-, and homozygous for the TA⁷  promoter allele at the UGT1A1 locus.

Findings in the relatives are shown in Table 1. The mother (I-1) is heterozygous for the triplicated α globin gene arrangement and the father (I-2) is both heterozygous for the same α globin gene arrangement and β thalassemia. Of the 4 siblings, II-2 is homozygous for the triplicated α globin gene arrangement, II-3 has a normal globin genotype, and II-4 has the same genotype as her father. The ^Gγ Xmn-1 and UGT1A1 genotypes of the relatives are summarized in Table 1.

The data shown in Table 2 are part of a much larger ongoing study of the genetic findings in patients with Hb E β thalassemia in Sri Lanka. For the purposes of comparison they show the Hb and Hb F levels, and ^Gγ Xmn-1 status. There are 29 patients with 4 α globin genes, 12 who are ^Gγ Xmn-1^+/+ and 17 who are ^Gγ Xmn-1^-/-.Two patients are also heterozygous for triplicated α globin genes, one ^Gγ Xmn-1^-/- and one Xmn-1^+/+. Clearly the ^Gγ Xmn-1 polymorphism has an important effect on the Hb F production in these patients, with a significantly lower level in those homozygous for the -/- arrangement. This effect is also seen in the patients who are heterozygous for triplicated α globin genes, but Hb F levels are in the range of those with 4 α globin genes.

For comparison, Table 2 also summarizes similar data from previously reported patients with extended α globin gene arrangements and from patients with milder forms of thalassemia intermedia as a result of a variety of genetic interactions.

The results of this study emphasize the complexity of genetic interactions that underlie the phenotype of β thalassemia intermedia and raise important questions about the regulation of Hb F production in the thalassemia syndromes.

Despite their disparate clinical treatment, the propositi in the 2 families described here show very similar hemoglobin analyses off transfusion, with marked anemia (Hb levels of 50-70 g/L [5-7 g/dL]) and very low levels of Hb F (3%-4%). As exemplified by the findings in the patients with Hb E β thalassemia, the degree of anemia is equivalent to those seen in severe β thalassemia intermedia associated with one severe and one less severe allele.¹¹  Analyses of α/β mRNA ratios demonstrate that the additional α genes are transcriptionally active, as in a previously reported αααα heterozygote with one β thalassemia allele.⁹  In the ααα/ααα β thalassemia homozygote, the high α/β mRNA levels were also consistent with increased output from the ααα chromosomes. The high α/β mRNA ratios are more extreme than the α/nonα globin synthesis ratios in the 2 propositi, probably reflecting reduced translation of the additional α globin mRNA.^18,19  However, the degree of globin chain imbalance, which might have been even higher if the incubation time had been less than 60 minutes, exceeds that seen in β thalassemia heterozygotes and is consistent with that found in the phenotype of β thalassemia intermedia.

It is generally considered that a large part of the increased Hb F in thalassemics is due to selective cell survival.¹¹  The cellular distribution of Hb F in adults is heterogeneous; any cells with excess α chains (whether because of a β thalassemia allele or additional α genes) that have increased γ chain synthesis should have reduced overall globin-chain imbalance and hence be more likely to survive in the bone marrow and to enter the circulation. This mechanism, together with a marked erythroid expansion, probably accounts for high levels of Hb F, although it is also believed that severe anemia alters the kinetics of erythropoiesis, predisposing red cell precursors to increase γ gene transcription, and that genetic factors may modulate the level.

The degree of globin-chain imbalance and the severity of anemia in mild or severe β thalassemia intermedia is normally associated with high Hb F, with levels commonly in the 20% to 80% range (Table 2). Yet in the propositi reported here, and in similar cases described previously, the Hb F levels are not significantly elevated (Table 2). What is the reason for this lack of Hb F response? Because the degree of globin-chain imbalance is similar, it is difficult to see why cells producing more γ chains in this condition should demonstrate any less selective survival than in any of the other forms of β thalassemia intermedia. Similarly, the presence of nucleated red cells in the peripheral blood, severe splenomegaly and markedly elevated Epo responses are compatible with both erythroid expansion and ineffective erythropoiesis.

The major difference between these cases and other forms of thalassemia intermedia is that there is only one defective β gene, whereas homozygotes for 2 mild β thalassemia alleles or compound heterozygotes for Hb E (a mild form of β thalassemia) and β thalassemia have deficient β chain production from both alleles. Table 2 compares the hemoglobin and Hb F levels in β thalassemia heterozygotes with additional α genes with similar data from other forms of β thalassemia intermedia, with or without additional α genes. Some of the variability in Hb F levels is determined by the C→T polymorphism at position -158 upstream of the ^Gγ gene (Xmn-1 - or +). However, even in the cases of Hb E/β thalassemia with 4 or 5 α globin genes, in whom the Xmn-1 status was -/-, the Hb F levels were significantly higher than in the β thalassemia heterozygotes with additional α genes. Furthermore, as shown in Table 2, very mild forms of β thalassemia intermedia because of homozygosity or compound heterozygosity for mild alleles have much higher Hb F levels than those heterozygous for β thalassemia with extended α globin gene arrangements. Although that action of other genetic modifiers of Hb F response may be responsible for producing the occasional unusually high level of Hb F observed within each of these forms of β thalassemia intermedia, given their low frequency, it is very unlikely that they could be responsible for the low levels of Hb F seen in those with extended α globin gene numbers.

It is possible, therefore, that for an adequate γ globin gene response to the effects of β thalassemia, mutations are required in both cis and trans. This would offer a unifying explanation for the very low levels of Hb F observed in relatively severe forms of β thalassemia in which there is only one defective β globin gene, that, as well as those with expanded α globin genes, include the dominant β thalassemias, also characterized by low Hb F levels.¹¹  If this were the case, it would require reappraisal of many of the issues regarding the regulation of Hb F production in adult life; therefore, this possibility requires further exploration. There are other possible explanations, including the relative degrees of erythroid expansion in these conditions. However, to reduce the level of Hb F to those seen in extended α gene interactions or dominant β thalassemia, the degree of expansion would have to be little more than that found in uncomplicated β thalassemia trait, a much milder disorder than those described here.

Much of the phenotypic difference between the propositi in these families can be ascribed to their clinical management. The propositus in Family H only received 1 unit of blood and has grown and developed normally at a hemoglobin value of approximately 50 to 70 g/L (5-7 g/dL). However, the propositus in Family G has been transfused regularly for many years without adequate chelation and is now dangerously iron loaded. This may well account for his growth retardation and lack of sexual development. Unlike the propositus in Family H, he is also deeply jaundiced in the absence of hepatic disease, a finding which he shares with several of his siblings. However, recent studies in Sri Lanka have shown that the homozygous inheritance of the 7/7 polymorphism of the promoter of the UGT1A1 gene is associated with severe jaundice in patients with Hb E β thalassemia.¹³  Presumably this is the basis for the unusual degree of jaundice associated with β thalassemia intermedia in this family.

Since the propositus in Family G has stopped blood transfusion and received intensive iron chelation, he has functioned extremely well at a hemoglobin level similar to the propositus in Family H. Because of the very low level of Hb F in these patients with triplicated or quadruplicated α globin genes it seems likely that they adapt unusually well to severe anemia compared with patients with β thalassemia intermedia with very high levels of Hb F, who cannot shift their oxygen dissociation curve to the right as an adaptive response.¹¹  Interestingly, current studies suggest that patients with Hb E thalassemia and similarly low Hb levels may even adapt to anemia in this way, despite their higher levels of Hb F, possibly as the result of the properties of Hb E.²⁰ 

Clinically, there is a very clear message from a comparison of the phenotypes of the 2 propositi in this study. It is vital to obtain a full genotype analysis and to carry out a long period of careful observation on growth and development in any infant with β thalassemia intermedia. Those with extended α globin genotypes, and in populations such as those of Sri Lanka they may not be uncommon, seem to grow and develop well even at low hemoglobin levels. As shown in Family G of the present study, their later problems may reflect their treatment rather than the disease itself.

We thank the members of families G and H for their cooperation, Amersham Biosciences, GE Healthcare for the gift of ³H leucine and help with its transportation, and Liz Rose for preparing this manuscript.