The Oxygen Affinity of Haemoglobin E

Oxygen dissociation studies were carried out on haemoglobin E (Hb E) at both high and low haemoglobin concentrations. Oxygen affinities of fresh red cells from three people homozygous for Hb E and from one with Hb E-beta thalassaemia (Hb-E trait/beta-thal trait) were low in three out of four patients studied, while the oxygen affinity of red cells from an individual with Hb-E was normal 2,3-DPG concentration in the fresh cells from the people with homozygous Hb E or Hb-E trait/beta-thal trait which showed low oxygen affinities were elevated sufficiently to account for the shifts observed. When the cells from two of these people with homozygous Hb E were depleted of 2,3-DPG. their oxygen affinities became the same as that of similarly treated normal cells. Pure 'stripped' Hb E in dilute solution behaved identically to Hb A in respect of P50, Bohr shift, haem-haem interaction, and interaction with inorganic phosphate or 2,3-DPG. Hb E, therefore, has the same oxypgen dissociation properties as Hb A both in dilute solution and in the red cell. The low oxygen affinities found in the fresh cells and in whole blood are caused by high 2,3-DPG concentrations within the cell.     

The instability of the membrane skeleton in thalassemic red blood cells

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta- thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha- globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.     

Oxygen dissociation curves in children with anemia and malignant disease

Two automatic apparatuses utilizing a dual wavelength spectrophotometer were used to perform oxygen dissociation curves on microsamples of blood. The method provides a complete print-out of an oxygen dissociation curve in 15--20 min and the P50 vlues obtained in normal individuals agree closely with those obtained by classical methods. These apparatuses were used to measure oxygen affinity, ie P50, in anemic children with malignant disease prior to treatment and in children undergoing therapy. Red cell 2,3-DPG levels were also measured. In patients with anemia at the time of diagnosis and prior to therapy, the P50 values and 2,3-DPG levels were elevated as is usual in other types of anemia. However, when oxygen affinity and 2,3-DPG levels were measured in anemic patients receiving treatment, three types of response to anemia were noted: 1) increased P50 and 2,3-DPG; 2) normal or low P50 and 2,3-DPG, and; 3) normal or low P50 with increased 2,3-DPG. Patients who adapted poorly to anemia during treatment had usually received prior intensive chemotherapy and transfusion therapy, and their pattern of red cell glycolytic intermediates was consistent with a red cell population with an increased cell age. The failure of some patients to respond to anemia with a decrease in oxygen affinity has implications in regards to the hemoglobin level at which they should be transfused.     

A novel α(0) -thalassemia deletion in a Greek patient with HbH disease and β-thalassemia trait

Objectives: To determine the molecular basis in a Greek child suspected of having HbH disease and β‐thalassemia trait. Methods: Standard hematology, Hb electrophoresis, and HPLC. Multiplex ligation‐dependent probe amplification (MLPA), direct sequencing, and breakpoint characterization by NimbleGen fine‐tiling array analysis. Results: The index patient showed a moderate microcytic hypochromic anemia with normal ZPP and elevated HbA₂, indicative for β‐thalassemia trait. However, the moderate microcytic hypochromic anemia along with the observation of HbH inclusions in occasional red blood cells suggested a coexisting α‐thalassemia. Molecular analysis indicated that the propositus inherited the β⁺‐thalassemia mutation IVS2‐745 (c>g) and a novel α⁰‐thalassemia deletion from the mother, and the common non‐deletion α‐thalassemia allele α₂(?5nt)α from the father. The α⁰‐thalassemia deletion, named ‐ ‐^BGS, is approximately 131.6 kb in length. It removes the major regulatory elements along with the functional α‐globin genes but leaves the theta‐gene intact. Conclusions: The compound interaction of a β‐thalassemia defect along with a single functional α‐globin gene is quite rare. Although patients with HbH/β‐thal and simple HbH disease have comparable levels of Hb, the absence of free β‐globin chains and thus detectable non‐functional HbH means that in HbH/β‐thal, the levels of functional Hb are higher, resulting in a better compensated functional anemia. Rare large deletions as the one described here remain undetected by gap‐PCR in routine molecular screening. The introduction of MLPA as a diagnostic screening tool may improve laboratory diagnostics for these defects. The use of NimbleGen fine‐tiling arrays may give additional information about the precise location of breakpoints.     

Diagnosis of concurrent hemoglobin H disease and heterozygous beta-thalassemia

Definitive diagnosis of concurrent hemoglobin (Hb) H disease and heterozygous beta-thalassemia cannot be made from Hb analysis alone, but necessitates genotype analysis and family study. Interactions between alpha- and beta-thalassemia must be considered when investigating moderate to severe hypochromic microcytic anemia of uncertain cause in adult patients from areas with a high prevalence of globin gene mutations.     

A rare example that coinheritance of a severe form of beta-thalassemia and alpha-thalassemia interact in a "synergistic" manner to balance the phenotype of classic thalassemic syndromes

The coinheritance of beta-thalassemia major with the genotype of Hb H disease is extremely rare, with few reported cases. We investigated the hematological, biochemical, biosynthetic, molecular and pathophysiological parameters to evaluate a rare male patient with this compound syndrome. The patient was studied at first diagnosis during hospitalization at 50 years of age and subsequently followed up for more than a year. Examinations included full hematological, biochemical, biosynthetic, molecular, pathophysiological and clinical parameters. Besides standard parameters, we additionally measured reticulocyte hemoglobin content (CHr), erythropoietin (Epo), soluble transferrin receptors (sTfR), oxygen pressure at 50% hemoglobin saturation (P50), 2,3-bisphosphoglycerate (2,3-BPG), total glutathione (GSHt), oxidized glutathione (GSSG), malonyldialdehyde (MDA), nontransferrin-bound iron (NTBI), vitamins A and E. The male patient was first hospitalized for a 2-day period at 50 years of age, following the finding of marked anemia (hematocrit 20%) during a blood test to investigate the cause of fatigue in the absence of weight-loss or other notable symptomatology. He had never been transfused, maintaining Hb 85-95 g/l. Definitive diagnosis was achieved through DNA studies, which showed coexistence of beta-thalassemia major (IVSI-6 T > C/IVSI-I G > A) with Hb H disease (-alpha(3.7)/-(Med)). Alpha/non-alpha globin chain biosynthesis was completely balanced. Parameters demonstrated a well-compensated anemia with ineffective erythropoiesis and oxidative stress, which was ameliorated following splenectomy. In conclusion, this case is a remarkable example that the coinheritance of severe forms of beta-thalassemia and alpha-thalassemia interact in a "synergistic" manner to almost complete balance the symptoms of classic thalassemia syndromes.     

Co-inherited β-thalassemia trait and HbH disease: clinical characteristics and interference in diagnosis of thalassemia by high-performance liquid chromatography

Introduction: To identify the clinical and hematological characteristics in a large group of patients with combined HbH disease and β‐thalassemia trait. Methods: Hemoglobinopathy analysis and full genotyping identified a cohort of patients with HbH disease, β‐thalassemia trait, or combined HbH disease and β‐thalassemia trait. Results: Co‐inheritance of β‐thalassemia trait and HbH disease significantly decreased the mean corpuscular volume (MCV) in 27 patients when compared to 287 patients with HbH disease alone. The combined condition also alleviated anemia in nondeletional HbH disease but not in the deletional cases. Beta‐thalassemia trait also significantly decreased the expression of HbH, Hb Constant Spring when present, and HbA₂, with levels as low as 3.6% on high‐performance liquid chromatography (HPLC). Conclusion: These cases, although relatively common in the South Chinese population, may be difficult do diagnose correctly when only examined on HPLC. Therefore, molecular analysis of the α and β globin genes should be done in all cases with hemolytic anemia and low MCV without clear HbH disease or β‐thalassemia parameters.     

Hb Dhonburi (Neapolis) [beta126(H4)Val-->Gly] identified in a family from northern Iran

Thalassemias are the most common hereditary diseases in Iran, resulting from synthesis defects in one or more hemoglobin (Hb) subunits. The majority of patients suffer from beta-thalassemia (thal), but cases with microcytic hypochromic anemia and normal electrophoretic patterns are suspected to have alpha- or silent beta-thal. A family from the northern part of Iran, an area highly prevalent for thalassemias, was referred to us for prenatal diagnosis. The hematological data of the father indicated a pattern of beta-thal minor. Reverse hybridization analysis for the most common beta-globin mutations identified IVS-II-1 (G-->A) in the heterozygous state. The maternal laboratory data indicated a case more compatible with alpha-thal. Iron deficient anemia was ruled out, and common alpha-thal point mutations and deletions were investigated. As no mutation was detected, chain synthesis was performed and showed an alpha/beta chain ratio of 2.1, that was in the range of beta-thal minor. DNA sequencing of the entire beta-globin gene identified a heterozygous GTG-->GGG (Val-->Gly) mutation at codon 126, also known as Hb Dhonburi (Neapolis). Prenatal diagnosis of the fetal DNA showed the absence of the IVS-II-1 and codon 126 anomalies. This result demonstrates the importance of screening of individuals with mild microcytic hypochromic anemia for both alpha- and silent beta-thal mutations.     

A family with segregating triplicated alpha globin loci and beta thalassemia

In this article we report a Sardinian family, in which a beta- thalassemia gene and a triple alpha-globin loci, counterpart of the rightward deletion type alpha-thalassemia-2, were segregating. The analysis of the genotype-phenotype correlations in the different family members allowed us to give an outline of the manifestations associated with different genotype combinations. The heterozygote for the triple alpha-loci showed no consistent abnormal clinical or hematologic characteristics and presented balanced alpha/beta-globin chain synthesis. In the homozygous state for this lesion, the only phenotypic expression was a slightly imbalanced globin chain synthesis. The combination of heterozygous beta-thalassemia with the heterozygous state for the triple alpha-globin loci produced no clinical manifestations and showed a hematologic phenotype indistinguishable from that of heterozygous beta-thalassemia. On the other hand, the combination of the homozygous state for the triple alpha-globin gene loci and the heterozygous state for beta-thalassemia produced a clinical picture of thalassemia intermedia with a very mild clinical course, minor increase of fetal hemoglobin (HbF) levels, and a pronounced imbalance of globin chain synthesis.     

A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in thai patients with thalassemia

The variety of patients with thalassemia in Thailand offers an opportunity to fully characterize the kinetic causes of the anemia and to study apoptosis of marrow erythroid precursors as a possible factor contributing to its severity. Kinetic studies showed that in hemoglobin H (HbH) disease, the extent of hemolysis, as well as the minimally ineffective erythropoiesis, usually falls within the compensatory capacity of normal erythropoiesis; therefore, anemia in patients with HbH partly represents a failure to expand erythropoiesis adequately. Hemoglobin Constant Spring (HbCS), a common variant of alpha thalassemia in Bangkok, causes more severe hemolysis and a distinct increase in ineffective erythropoiesis. Ineffective erythropoiesis plays a much more prominent role in beta thalassemia/hemoglobin E (beta-thal/HbE) disease, in which the variability of the anemia is puzzling. We compared mild and severe cases and found that patients with severe disease had a maximal marrow erythropoietic response that failed to compensate for very short survival of red blood cells and a marked quantitative increase in ineffective erythropoiesis. Analysis of apoptosis of marrow erythroid precursors done both on shipped samples and in Bangkok showed a moderate increase in HbH disease, consistent with the small increase in ineffective erythropoiesis. In patients with homozygous HbCS, there was a further increase in apoptosis, consistent with the additional increase in ineffective erythropoiesis. Patients with beta-thal/HbE disease had the most ineffective erythropoiesis and the most erythroid apoptosis. Thus, it appears that alpha-chain deposition in erythroid precursors, either alpha(A) or alpha(cs), leads to accelerated apoptosis and ineffective erythropoiesis.     

Preliminary data on preimplantation genetic diagnosis for hemoglobinopathies in Turkey

Hemoglobinopathies are the most common genetic diseases in Turkey. Sickle cell anemia is prevalent in the Cukurova region but beta-thalassemia (thal) is seen all over the country. The incidence of sickle cell trait is 10% and beta-thal trait is 3.7% in this region. The families at risk for hemoglobinopathies have come to our center for prenatal diagnosis since 1992. In 15 years, 1,593 fetuses were examined. Four hundred and ten fetuses were found to be homozygous or compound heterozygous for sickle cell anemia and beta-thal. Some mothers had affected fetuses several times. Preimplantation genetic diagnosis (PGD) is an option to avoid the termination of a pregnancy. Studies for PGD of sickle cell anemia were done at Cukurova University Hospital, Cukurova, Adana, Turkey.     

Increased circulating activated endothelial cells,vascular endothelial growth factor,and tumor necrosis factor in thalassemia

An increased number of circulating endothelial cells (CECs) was demonstrated in alpha- and beta-thalassemic patients, beta-thalassemia/hemoglobin E (BE), both splenectomized (BE[S]) and non-splenectomized (BE[NS]), had higher numbers of CECs than alpha-thalassemia, both HbH (alpha-thal l/alpha-thal 2; H) and HbH with hemoglobin Constant Spring (alpha-thal 1/CS; H/CS). CECs were also increased in heterozygous HbE (EA) and homozygous HbE (EE). The highest level of tumor necrosis factor-alpha (TNF-alpha) was found in HbH/CS patients, whereas the highest levels of vascular endothelial growth factor (VEGF) was observed in BE[S] patients. Significant decreases, in protein C and protein S levels were found in both alpha- and beta-thalassemia compared with normal. Good correlations between the numbers of CEC and TNF-alpha, VEGF, protein C, and protein S levels were demonstrated in this study. In addition, markers for endothelial cell activation and injury (intercellular adhesion molecule-1, ICAM-1/CD54; vascular cell adhesion molecule-1, VCAM-1/CD106; and E-selectin, ELAM-1/CD62E) were detected on the surface of isolated CECs using immunofluorescence technique. Appearance of CECs with markers for endothelial cell activation, together with increased levels of TNF-alpha and VEGF and decreased levels of protein C and protein S in the circulation, may account for the propensity of vascular perturbation in thalassemic subjects.     

Control of oxygen affinity of hemoglobin in K562 cells induced by hemin

The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.     

In Vivo Aging of Transfused Erythrocytes and 2,3-Diphosphoglycerate Levels

Levels of 2,3-diphosphoglycerate (2,3-DPG)and oxygen affinity were measured in apatient with refractory congenital hypoplastic anemia in order to determine theresponse of aging transfused erythrocytesto a decrease in tissue oxygen tension.Ferrokinetic studies revealed that the rateof erythropoiesis after transfusion wasapproximately 10% of normal. In the interval between transfusions, the level of2,3-DPG and P₅₀ rose to values considerably above the normal range as the levelof the hematocrit decreased. On fiveseparate occasions, the 2,3-DPG concentration was elevated (average 25.5 µmoles/gof hemoglobin) and the P₅₀ level increased(average 33.2 mm of mercury) 24 hr priorto transfusion. It is concluded that in vivoaging of transfused erythrocytes does notprevent these cells from responding to anincreasingly hypoxic environment by elevating 2,3-DPG levels and by decreasingoxygen affinity.

Submitted on August 10, 1972 Revised on November 6, 1972 Accepted on November 18, 1972     

A new sickle cell disease phenotype associating Hb S trait,severe pyruvate kinase deficiency (PK Conakry), and an α2 globin gene variant (Hb Conakry)

A Guinean woman, hetererozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C → A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [α80(F1) Leu → Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n_max value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.     

Red Cell 2,3-Diphosphoglycerate Levels in Children with Hereditary Haemolytic Anaemias

The role of red cell 2,3-diphosphoglycerate (2,3-DPG) in increasing the availability of haemoglobin oxygen in neonatal jaundice and hereditary haemolytic anaemias was investigated. Measurements of 2,3-DPG were carried out on 58 normal children and six normal adults, 18 full-term newborns with neonatal jaundice and 57 cases (51 children and six adults) with hereditary haemolytic anaemias. In normal children and adults, with a mean haemoglobin of 12.69 g/dl, mean 2,3-DPG was 14.90 mumol/g Hb. In jaundiced newborns with a mean haemoglobin of 16.04 g/dl mean 2,3-DPG levels were 14.51 mumol/g Hb, i.e. normal. 2,3-DPG levels were increased in patients with beta-thalassaemia major, alpha-thalassaemia, sickle-cell disease, favism, hereditary spherocytosis and in heterozygotes for beta-thalassaemia with increased haemoglobin F. In heterozygotes for beta-thalassaemia with increased haemoglobin A2 only and in sickle cell trait 2,3-DPG levels were normal.     

Globin chain synthesis is a useful complementary tool in the differential diagnosis of thalassemias

The present study aimed at differentiating rare types of heterozygous beta-thalassemia (thal) with normal Hb A(2) values from alpha-thal in Iranian carriers by globin chain synthesis in addition to other hematological parameters. Our study groups consisted of 51 normal subjects, 24 heterozygous beta- thalassemic subjects with high Hb A(2), 62 alpha-thal-2 subjects, 34 alpha-thal-1 subjects, six Hb H disease thalassemic subjects, 14 silent beta-thal subjects with normal Hb A(2) values, five deltabeta-thal subjects and two subjects with an association of alpha- and deltabeta-thal (total = 198). Analysis of globin chains was performed by high performance liquid chromatography (HPLC). The results showed that the alpha/beta ratio averages were close to the ones in the published literature, but with a greater standard deviation and a wider range. Globin chain synthesis (GCS) could be valuable in differentiating between microcytosis produced by silent beta-thal (heterozygous beta-thal with a normal Hb A(2) level) and that caused by alpha-thal. Since the complex genotype/phenotype relationship can lead to diagnostic difficulties, GCS cannot be used as the only diagnostic tool for thalassemia carrier detection. Therefore, a combination of different tests for each patient is required.     

beta-Thalassemia arising as a new mutation in an American child

A 6-year-old child of northern European ancestry was found to have microcytic, hypochromic anemia with an elevated level of hemoglobin A2 and an unbalanced pattern of globin chain synthesis characteristic of beta-thalassemia trait. Hematologic and globin synthesis studies of both parents yielded entirely normal results. Identification of the mother and father as the biological parents was established with a high order of reliability by determination of erythrocyte, serum, and HLA genetic markers. These findings suggest that the picture of beta-thalassemia observed in this child represents a new mutation.     

The gamma chain heterogeneity of fetal hemoglobin in black beta- thalassemia and HPFH heterozygotes

High pressure liquid chromatography (HPLC) was applied to the HbF isolated from blood of numerous black patients with beta-thalassemia trait or homozygosity, G gamma-delta beta-thalassemia trait, G gamma A- gamma HPFH heterozygosity, or the G gamma-[delta+ beta+]-HPFH condition. The method allowed an accurate evaluation of the relative quantities of three types of gamma-chain (G gamma, A gamma I, A gamma T) in the fetal hemoglobins. The results have shown the following. (A) The incidence of the A gamma T-chain in beta-thal heterozygotes and G gamma A gamma-HPFH heterozygotes is about the same as has been observed in black newborn; about one of five blacks are heterozygous for this A gamma-chain variant. The A gamma T-chain was not detected in the nine G gamma-delta beta-thal heterozygotes nor in the eight G gamma-[delta+ beta+]-HPFH heterozygotes. (B) In most cases, the A gamma T-chain was produced by the A gamma gene in trans to the beta-thal or HPFH determinant. The contribution by the gamma-chain genes in trans to the beta-thal or HPFH determinant is about 15% of the total gamma-chain production in both conditions. (C) Three black beta-thal heterozygotes (and five additional relatives) had the A gamma T gene in cis to the beta-thal determinant. Four of these patients had a low levels of G gamma-chain (the "adult" level), and the contribution by the A gamma gene in cis to the beta-thal determinant was about three times that of the A gamma gene in trans. The four additional patients, all members of one family, had a high level of G gamma-chain (the "newborn" level), and the contribution of the A gamma gene in cis was half of that seen in the previously mentioned four patients while that of the A gamma gene in trans was essentially the same. These limited data suggest that the genetic anomaly causing high high G gamma levels in adult beta-thal heterozygotes is linked to the beta-thal determinant and that one of its primary effects is a decreased synthetic expression of the A gamma gene in cis to the beta-thal determinant.