Search

Reversed phase high performance liquid chromatography of globin chains is an important additional tool in the study of hemoglobin abnormalities. Using a technique modified from that of Leone et al.,[1] we report here the relative chromatographic behavior of about 200 different hemoglobin variants. This method provides an additional dimension in the presumptive characterization of hemoglobin variants. It was also found to be of special value for measuring the expression of neutral variants, such as thalassemic or unstable hemoglobins, and to identify neutral mutations associated with another variant, resulting in unusual hematological presentations.

Hb J Sardegna is a well known innocent Hb variant which is widespread in Sardinia. As yet, homozygosity for Hb J Sardegna has not been documented. This report deals with the homozygous state for Hb J which we demonstrate by molecular analysis in two Sardinian siblings in which b-thalassemia coexists. The Hb J specific mutation was determined both by enzyme digestion and by sequencing specific segments of PCR amplified a-globin genes. A pregnant girl showed mild non-sideropenic microcytic anemia, normal Hb A2 levels (2.4%) on DE-52 microchromatography, 50% of Hb variant on HPLC and 2.1 a/b globin chain biosynthetic ratio. She proved to be a carrier of the b86(-A) thalassemia determinant. The aglobin gene mapping did not reveal a-thalassemia. Btg I restriction analysis of both a2-globin genes showed a recognition site defect for this enzyme in both chromosomes, which resulted to be the C?A point mutation in homozygosity at the first nt of a2-globin gene 50th codon by sequencing. This defect, typical of Hb J Sardegna, was also present in her brother. From a practical point of view, this study demonstrates that the association of b-thalassemia with Hb J, may show falsely reduced Hb A2 levels on routine Hb A2 quantitation techniques, such as DE-52 microchromatography. This possibility implies that identification methods such as simple Hb electrophoresis, which permit visualization of Hb A J 2 should be used in thalassemia screening involving populations in which Hb J and b-thalassemia coexist. The Hematology Journal (2002) 3, 176‐178. doi:10.1038/sj.thj.6200172

The influence of repeated hypoxia on the development of haemoglobin (Hb) subtypes and on extramedullary haematopoiesis (EMH) was investigated in young Wistar rats of different ages. The rats were exposed to hypercapnic/hypoxic and to "simple" hypoxic conditions. The results obtained were compared to those of an untreated age-matched control group. Different globin chains were measured using HPLC and time-of-flight (TOF) mass analysis. The number of EMH cells was evaluated by cell counting. By determining the proportions of alpha- and beta-chains, fetal, neonatal and mature types of globin chain composition could be differentiated. The beta-2 chain levels were significantly higher in hypercapnic/hypoxic environments than in the controls and simple hypoxic environments. The numbers of EMH cells in the two groups subjected to hypercapnia/hypoxia decreased significantly more slowly compared to the controls and simple hypoxia groups. Therefore, the development of Hb subtypes and the EMH activity in rats were influenced by both repeated hypercapnia and hypoxia.

Advancements in the understanding of the hemoglobin molecule and its variants have been a driving force for medical and molecular genetics, making hemoglobin one of the best-characterized human proteins. These achievements in discovery and comprehension are indebted to the interplay between clinical and laboratory observations, to the abundance of nonlethal hemoglobin variants, and to the relative ease of obtaining large amounts of protein by simple venipuncture. As always, the productive application of science lags behind the discovery itself; hemoglobinopathies and thalassemia syndromes are no exception. However, each passing decade has held developments that have changed the natural history of these disorders.

Using Brownian motion simulations we have studied the formation of docked complexes of reduced cytochrome b5 and oxidized haemoglobin. Our results indicate that the presence of molecular electrostatic fields has a significant role to play in the formation of these complexes. In contrast to previous modeling studies on this system, we clearly identify electron transfer within an ensemble of similarly docked complexes rather than the formation of a single complex. Docking involves a number of acidic residues surrounding the exposed haem edge of cytochrome b5 and a set of basic residues surrounding the exposed haem edge of the globins. Although amino acids from the partner globin proteins are involved to a small extent in the binding of some of the complexes, the reactivity of any particular globin is essentially independent of the nature of its partner globin chain within the haemoglobin molecule. Comparison of results from adult and embryonic haemoglobins indicates a significant difference in complex formation. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes. These data provide a theoretical insight into the important process of re-reduction of oxidized haemoglobins as well as explaining the general inability to produce crystalline forms of many docked electron transfer complexes.

Comparative studies of haemoglobin (Hb) function are of special interest since they may lead to an understanding of those changes which, in the course of evolution, have developed in different organisms to meet specific physiological requirements.

The hemoglobinopathies belong to a diverse group of inherited disorders characterized by the reduced synthesis of one or more globin chains (thalassemia) or the synthesis of a structurally abnormal hemoglobin (Hb). Approximately 900 different hemoglobin variants characterized by mutations involving alpha, beta, gamma, and delta globin chains have been described worldwide. In Brazil, the high degree of ethnic admixture among native Americans and African and European descendants has produced elevated frequencies of Hb alterations. The aim of the present study was to characterize globin chain mutants based on classical laboratory tests and molecular analyses to supply detailed information about the Hb diseases to health professionals and to contribute to the knowledge of abnormal hemoglobins in Brazil.

AIMS: To document current United Kingdom practice for antenatal screening for inherited disorders of globin chain synthesis and to compare such practice with guidelines published by the British Committee for Standards in Haematology and the Standing Committee on Sickle Cell, Thalassaemia and other Haemoglobinopathies (SMAC). METHODS: The members of the UK Forum on Haemoglobin Disorders were surveyed about their current practice for antenatal haemoglobinopathy screening. The UK Forum is a national group of haematologists, paediatricians, laboratory scientists, and counsellors working in the field of diagnosis and management of disorders of haemoglobin synthesis; such disorders including the alpha and beta thalassaemias, sickle cell disease, and other haemoglobinopathies. RESULTS: Completed questionnaires from 38 hospitals (or cooperating groups of hospitals) were analysed. The great majority of hospitals were applying appropriate laboratory methods, but problems were commonly encountered in ensuring that appropriate testing of antenatal patients and, when necessary, of their partners, was carried out early in pregnancy. When screening was selective there was quite often a failure to identify all women in whom testing was indicated, and cut off points used as an indication for further testing were sometimes inappropriate. CONCLUSIONS: Many practical problems are still encountered in following guidelines for the antenatal diagnosis of haemoglobinopathies. A need for improved administrative procedures and increased funding was identified. In addition there is a need for agreed guidelines giving more specific advice on technical aspects of laboratory practice.

All human hemoglobins have a tetrameric structure, consisting of two identical α-like (α or ζ) and two β-like (e, γ, δ, or β) globin chains, each linked to a heme group.

Publisher Summary This chapter presents the structural characterization of chemically or genetically altered hemoglobins at the level of modifications to the primary structure. Most of the procedures used depend on high-performance liquid chromatography (HPLC). Strategies for structural characterization necessitate several steps before peptide and amino acid analyses can be done because chemical modifications often lead to heterogeneous mixtures of reaction products. The chapter presents a scheme for structural characterization of modified hemoglobins and outlines the steps utilized. The first step is to assess the degree of heterogeneity of the material to be characterized. The second step, if possible and practical, should be the isolation of single pure hemoglobin components by either preparative ion-exchange HPLC or low-pressure liquid column chromatography. The third step is to assess heterogeneity of the reaction mixture and to identify the modified globin chain(s) because of the presence of two different kinds of globin chain subunits in most hemoglobins. This is most simply and rapidly accomplished by analytical reversed-phase HPLC using a C4 column. The fourth step is the actual isolation of the modified globin chain of interest by preparative reversed-phase HPLC.

About 150 million people throughout the world carry abnormal hemoglobins, with consequences ranging from trivial to lethal. More than one in 800 people have hemoglobins that differ from normal by amino acid substitutions, globin chain elongations, contractions, and fusions. In determining this incidence, the most common ones—variants Hb S, Hb E, and Hb C—were not included (1).

Summary A rather common haemoglobin variant was detected in the Sardinian and Altamurana sheep breeds. The mutated globin chain appears to be produced under the control of an allele at the HBB locus and due to a neutral amino acid substitution. The variant will be provisionally referred to as the Hb I.

A procedure for structural analysis of normal and variant human hemoglobins by using fast atom bombardment mass spectrometry is described. The methodology consists of direct mass spectrometric analysis of proteorytic digests of the variant globin; peptide recognition along the globin chain sequence is accomplished on the basis of their molecular weight. The abnormal peptide(s) will exhibit a mass value different from those of the normal hemoglobin, allowing easy definition of the structural variations. The application of this procedure to the determination of normal globin chains as well as to known and unknown abnormal human hemoglobins is reviewed.

(1992). HB Kaohsiung or New York: A T→A Substitution at Codon 113 of the β-Globin Chain Creates an Alu i Cutting Site. Hemoglobin: Vol. 16, No. 1-2, pp. 123-125.

Globin chain synthesis was studied in the reticulocytes of 30 patients with various myelodysplastic syndromes (MDS) to determine the alpha:beta globin chain synthetic ratio and its probable prognostic value. The mean (SD) value of the total alpha:beta ratio was 0.82 (0.45) ranging from 0.05 to 1.73. The same ratio in 10 normal controls was 1.01 (0.04). This difference was significant. Furthermore, the alpha:beta ratios were lower than normal in 14 patients (alpha-thalassaemia-like) (group I), almost within normal limits in 11 (group II), and higher than normal in five (beta-thalassaemia-like) (group III). In each group almost all the FAB subtypes were represented. The addition of exogenous haem in several of the test samples resulted in a slight to pronounced increase in the alpha:beta ratios, particularly in group I. In 92% of the high risk cases (refractory anaemia with excess blasts (RAEB), chronic myelomonocytic leukaemia (CMML] or 87.5% of patients who finally developed acute non-lyphoid leukaemia (ANLL) low or normal alpha:beta ratios were found. No significant correlation was noticed between alpha:beta ratios and various haematological variables or survival. It is concluded that in MDS the alpha:beta ratio varied enormously across the entire population of patients, as well as within each FAB subtype, thereby restricting its prognostic value. Although haem deficiency may be implicated in some cases of MDS, why this should be remains unclear.

The crystal structures of the Hbs from the clam Scapharca inaequivalvis (Hbl, a homodimer, and Hbll, a heterotetramer) (1-3), and from the “fat innkeeper” worm Urechis caupo (a homotetramer) (4) have revealed novel assemblages of Mb-folded chains that differ markedly from that characteristic of the α2β2 vertebrate Hb tetramer (5).

We carried out a hemoglobin (Hb) survey on 7,250 blood samples from healthy donors. The routine analysis of Hb was done by electories, Beaumont, TX, U.S.A.) (1). Further identification of variant Hbs was made by isoelectrofocusing (IEF) on thin-layer polyacrylamide gels at pH 6.0-9.0 (2), and globin chain electrophoresis on cellulose acetate strips at pH 8.5 and 6.0 (3,4), with comigration of reference samples of Hbs S, D, and C

To the Editor: The clinical evaluation of treatments for disorders of globin-chain synthesis1–4 has entered an exciting new era. Decisions about the design and application of future trials will dra...

This symposium is concerned with recent studies that have attempted to define the molecular basis of the defects in globin synthesis which produce the hematologic manifestation of thalassemia. There have been two basic approaches to this problem. Analysis of the structure and synthesis of the various mutant globin chains in patients has permitted the formulation of a genetic hypothesis by which deletion of specific structural or regulator genes could produce the various forms the thalassemia. The second approach involves direct search for defects in the biochemical processes by which each structural gene directs the synthesis of a specific globin chain. The recent developments of these studies are reviewed and discussed briefly.

The presence of Hb New York was confirmed in a Chinese family in which affected members have occasional red cells with Hb-H-like inclusions and a relative decrease in alpha chain synthesis, suggestive of a coexisting alpha thalassaemia trait. However, globin gene mapping and DNA hybridization revealed no deletion of the alpha genome. Timed-incubation experiments showed that the rate of synthesis of beta NY chain was greater than that of normal beta chain in the early periods. Chromatographic separation of Hb NY and Hb A before chain analysis revealed preferential binding of newly synthesized alpha chains to beta NY, with a four-fold increase in specific activity of the alpha Hb NY chains. It is concluded that beta NY chain is being synthesized more rapidly and its increased turnover may account for this presentation of apparent alpha chain deficiency.

An unusual family is described with a congenital bleeding disorder present in four males belonging to three generations. Of the three surviving affected males, all had splenomegaly and petechiae. The three had moderate thrombocytopenia (55–90 X 10(9)/liter) and markedly prolonged Ivy-template bleeding times (greater than 30 min). They were also noted to have reticulocytosis and, upon further investigation, imbalanced globin chain synthesis resembling that of beta-thalassemia minor. Studies on nine additional family members in four generations were normal except for slight elevations of reticulocyte counts in female members, one of whom had the abnormal globin chain synthesis ratio. In male members, the bleeding tendency and clinical signs always occurred in the presence of the globin chain synthesis defect and reticulocytosis. This previously undescribed condition was apparently transmitted as an X-linked disorder.

A simple isoelectric focusing technique is presented for the screening of alpha- and beta-globin chain variants on polyacrylamide gel in the presence of 8 M urea. No detergent was used. Variants focused at discrete pH values, depending on the charge variation (-2, -1, 0, +1 or +2) introduced by the mutation. A linear relationship was evidenced between the charge variation and the shift of the apparent isoelectric point of the variants. The practical interest of this procedure is illustrated.

A simple and rapid micropreparative method for isolating 5 to 30 nmol of globin chain, followed by structural identification of abnormal human hemoglobins, is described. The method is based on the electrophoretic separation of globins on ordinary Cellogel films under denaturating conditions with subsequent cutting out of the protein zones and solubilization of Cellogel and the electrophoretic buffer components in a specially selected solvent in which the globin chain undergoes quantitative precipitation. The method makes it possible to simplify and speed up the structural identification of commonly occurring abnormal hemoglobins. The advantages and limitations of the method are discussed along with its potential uses in structural protein chemistry.

The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.

Two sons of a previously reported Ghanaian homozygote for the hereditary persistence of fetal hemoglobin (HPFH) (Ringelhann et al., 1970) also are HPFH homozygotes. In addition, another unrelated adult Ghanaian homozygote has been detected. All of these Ghanaian homozygotes as well as three American Black HPFH homozygotes have the G gamma A gamma type of HPFH with a G gamma to A gamma ratio of about 3:2, in contrast to an Asiatic Indian homozygote who has the G gamma type. Globin chain synthesis in HPFH homozygotes is unbalanced, with a gamma/alpha ratio of 0.6 or less, whereas it is balanced in heterozygotes according to most reports.

Summary Reticulocytes were isolated from the blood of eight nonanemic individuals with sickle cell trait; five donors had relatively low hemoglobin S concentrations (26 to 32%) and three had high hemoglobin S concentrations (40 to 42%). The cells were incubated with [ 3 H]leucine in a medium supporting protein synthesis. Total α chain synthesis was equal to total β chain synthesis in cells from the high hemoglobin S donors. In cells from low hemoglobin S donors the rate of α chain synthesis was about 65 to 80% that of total β chain synthesis. These data suggest that there was a deficit in α chain synthesis in the cells of sickle cell trait individuals with low hemoglobin S concentrations. The results are consistent with the presence of an α-thalassemia or α-thalassemia-like gene in the low hemoglobin S people.

The synthesis of hemoglobin alpha-chains was investigated in splenic colonies derived from syngenic colony-forming units injected in lethally irradiated C3H mice. Whereas normal adult mice of this strain synthesize two structurally distinct alpha-chains, the "alphaC57" and the "alpha NB", chains, it was found that erythrocytes from splenic colonies of irradiated recipient mice contained no alpha chain of the C57 type. Daily injection of serum from adult C3H mice into irradiated recipient mice of the same strain stimulates the synthesis of alphaC57 chain, whereas the injection of serum of lethally irradiated mice, drawn 8 days after irradiation, does not. It is concluded that the serum of adult C3H mice contains one or several factors that are responsible for the differential activation of the synthesis of the alphaC57 globin chain. This factor is shown to be associated exclusively with a high molecular weight fraction of serum.

Two families from southern India with members having the clinical manifestations of thalassemia intermedia are presented. Hematologic and globin chain synthesis data indicated that in one family the a

The factors responsible for the switch from gamma- to beta-globin synthesis, which occurs in man in the perinatal period, are still unknown. In this study the relationship of fetal maturity (as measured by gestation age and fetal weight) to changing rates of globin synthesis was analyzed. The relationship of rates of erythropoiesis to the switch was also analyzed. Rates of globin chain synthesis were studied in blood from fetuses from 10-41 wk gestation, in neonates up to 6 mo of age, and in a series of twins from 29-40 wk gestation. The beta/gamma-globin synthesis ratio in fetal blood remained relatively constant until 30-34 wk gestation when an increase in beta-globin production was observed. Adult levels of globin synthesis were present in neonates at 6-9 wk after birth. The gamma/alpha-globin synthesis ratio showed a reciprocal change, falling after 30 wk gestation. Globin synthesis ratios in fetuses and neonates from 32-41 wk post-conception showed a significant correlation with age (beta/gamma: p less than 0.001; beta/alpha:p less than 0.001; gamma/alpha: p less than 0.05), and there were significant correlations between the beta/alpha and beta/gamma synthesis ratios and weight (p less than 0.05 for both ratios). Globin synthesis ratios in 12 sets of twins from 29-40 wk gestation were generally within the normal range for gestational age. However, the switch to beta-globin production was more advanced than expected from weight for some of these babies.

A 25 year old woman with congenital dyserythropoietic anemia (CDA) Type I is described. Typical morphologic abnormalities of the erytliroid precursors in the bone marrow by light and electron microscopy, marked ineffective erythropolesis and iron loading were present. Globin chain synthetic ratios as well as functional and structural studies on the patient's hemoglobin were normal, ruling out the presence of thalassemia or a mutant hemoglobin which can both give rise to morphologic and clinical features similar to CDA. The laboratory findings on this patient and family members and a brief review of the literature are presented.

SUMMARY. Globin chain synthesis was studied in seven severely iron-deficient patients before and after treatment with iron. There was no appreciable difference between the individual pre- and post-treatment a/P specific activity ratios and the mean a/P ratio for each group was 1*00+SD 044. In a further six untreated iron-deficient patients the mean alp ratio was 1-00? SD 0.04. There was therefore no evidence that iron deficiency caused a reduction in alp ratio. Three patients with p thalassaemia trait and coexistent iron deficiency had lower alp ratios before treatment than after treatment with iron. It appeared that iron deficiency had caused reduced a chain synthesis in this groap. Preliminary experiments have shown that the alp specific activity ratio of purified 1i.icmoglobin A is decreased in iron deficiency, indicating an increase in the size of the free a chain pool. It is suggested that iron deficiency may interfere with the proteolytic mechanism normally responsible for the destruction of excess a chains. In combined iron deficiency and p thalassaemia trait, the resulting increase in free a chains might act by negative feedback to inhibit further a chain synthesis (Blum et a\, 1970) thereby reducing the pre-treatment alp ratio. The essential precursors involved in haemoglobin synthesis are iron, porphyrins and globin, with ferrous iron and protoporphyrin combining to form the molecule of haem. In iron deficiency, reduction in haem synthesis results in severe impairment of haemoglobin production. Although reduced haem synthesis is the major factor responsible for the microcytic hypochromic anaemia of iron deficiency, there remains the possibility that globin chain synthcsis may also be affected. It has been claimed (White et al, 1971; Ben-Bassat et al, 1974) that iron deficiency may cause a reduced rate of synthesis of the a chain of globin relative to that of the p chain. Biosynthetically, iron deficiency would then be indistinguishable from a thalassaemia trait, a disorder characterized by a reduced rate of a chain production (Kan et all 1969). If this were so, the usefulness of globin chain biosynthetic studies in the differential diagnosis of refractory microcytosis and in the positive identification of random carriers of a thalassaemia trait (Walford &. Deacon, 1976) would be seriously undermined.

In this study the clinical and hematological characteristics, the transmission pattern and the relative rates of globin chain synthesis were determined in the members of four Sardinian families with 14 patients affected by hemoglobin H disease. The severity of hemoglobin H disease in Sardinian subjects shows a high degree of variability. Clinically it usually appears intermediate between the hemoglobin H disease found in Oriental and Negro populations. The alpha/beta specific activity ratio was 0.42 +/- 0.10 indicating an analogous biochemical defect like that described in the Chinese. On the basis of hematological data and alpha/beta ratio, the genetics of hemoglobin H disease in Sardinians seem to follow a pattern similar to that observed in Orientals: one parent showing alpha-thalassemia-1 trait and the other alpha-thalassemia-2 trait. Parent offspring transmission of hemoglobin H disease did occur in 2 out of 6 hemoglobin H matings with spouses carrying the alpha-thalassemia-1 gene. This observation indicates either a high frequency of alpha-thalassemia trait in Sardinians or a high incidence of inbreeding. In one family the mating of a patient with hemoglobin H disease and a normal person produces 6/6 offspring with alpha-thalassemia-1. The genetic implications of this transmission pattern are discussed.

Determination of the biosynthetic beta/gamma ratio in samples of fetal blood is used for prenatal diagnosis of thalassemia. The current method, carboxymethyl cellulose chromatography (CMC), is cumbersome, slow, and expensive. Radiolabelled globin chains (A gamma, G gamma, beta, and alpha) can also be separated by electrophoresis in slab gels containing polyacrylamide, acid, urea, and Triton X-100. The radioactive bands are detected by fluorography. We determined the beta/gamma ratio in 24 samples of blood from fetuses at risk for beta thalassemia. CMC column data were compared with quantitations from fluorograms of slab gels. The beta/gamma synthetic ratios correlated (r=0.91), although the ratios were slightly less by gel than by column. The gel method is simple, inexpensive, and permits up to a dozen simultaneous analyses. Confirmatory CMC columns need only be run if the beta/gamma ratio is between 0.01 and 0.03 by gel.

3 cases of thalassemia intermedia have been found in the same family. The parents are not consanguineous but both come from the same town of Calabria (Italia). The mother is a heterozygote for β-thalassemia, as well as the father whose globin chain synthesis is nevertheless balanced, thus suggesting an association with α-thalassemia. This hypothesis is confirmed by the fact that one of the offspring shows the typical characteristics of α-thalassemia heterozygosity. The 3 subjects with thalassemia intermedia are synthesizing the β+-globin chain in a proportion higher than that expected from the level of Hb A in peripheral blood. In 2 of them, the globin chain biosynthetic ratio measured in the blood reticulocytes is not significantly different from that usually observed in thalassemia major of either the β° or β+type. In the third subject the globin chain synthesis is slightly less unbalanced probably because an α-thalassemia is also present. This suggests that factors other than a lesser imbalance in globin chain synthesis are involved in the occurrence of thalassemia intermedia. One of these factors could be a better survival of cells richer in Hb F than in Hb A, since these cells must have a lesser excess of α-chains.

Globin chain synthesis has been investigated for the first time in 2 patients homozygous for haemoglobin Lepore, although the 2 brothers have the same haemoglobin genotype the severity of the diseases is very different. The purpose of this study was to try and find out the reason for the different severity in the clinical manifestations. In the 2 patients a different excess of alpha-chain synthesis was observed, the higher excess being present in the subject carrying the more severe anaemia. This result strongly suggests that in homozygous haemoglobin Lepore disease, as in beta-thalassaemia, the degree of globin chain imbalance is responsible for the clinical manifestations.

An abnormal, fast-moving haemoglobin was observed in 5 healthy subjects of a family from Calabria (southern Italy). In all these carriers the abnormal haemoglobin, which structural studies identified as Hb Norfolk (α257(E6) GIy→Aspβ2) [4], occurs at a level averaging 33% of the total haemoglobin. Biosynthetic studies showed no evidence for unbalance of the globin chain synthetic ratio. In order to account for the observed percentages of Hb Norfolk, current concepts about the α-globin chain genetic system are reviewed, and different genie arrangements which would be in agreement with the experimental findings are discussed.

To be useful in the study of mechanisms controlling hemoglobin synthesis, erythroid bursts cloned from peripheral blood burst-forming units (BFU-E) must be shown to perform functions characteristic of bone marrow erythroid cells. To this end, peripheral blood BFU-E were cloned from two carriers of Hb Lepore. This globin chain has the N-terminal sequence of the δ chain and C-terminal sequence of the β chain. Reticulocytes from carriers of Hb Lepore do not synthesize the δβ chain and also synthesize an excess of α chain. In contrast, nucleated erythroid cells from bone marrow of Hb Lepore carriers can both synthesize the δβ chain and maintain balanced synthesis of the α and non-α chains. Like bone marrow cells, colonies derived from BFU-E of the Hb Lepore carriers incorporated 3H-leucine into a peak that cochromatographed with the δβ chain and contained 9%–16% of the non-α chain counts. Colonies derived from peripheral blood BFU-E from normal persons incorporated little radioactivity into this position. The counts in the δβ position were unlikely to represent artifacts of the culture system, since little radioactivity was found in this position when 3H-isoleucine was used as a label. When hemoglobins A2, Lepore, and A + F were isolated before chain separation, both the δ and the δβ chains contained significant amounts of radioactivity. Neither of these chains was synthesized in reticulocytes from either subject. Thus, the cultured cells, like bone marrow cells, must synthesize and translate the mRNA for each of these globin chains. In addition, cultured cells also behaved like bone marrow cells in that an excess of radioactive α chains was not observed, suggesting that the cultured cells possess the balancing mechanisms found in bone marrow cells.

Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia has been studied. A good correlation between an electrophoretic and a Chromatographie method on carboxymethylcellulose was found and the diagnoses suggested by both methods were always coincident. The electrophoretic method was also utilized for HbS/β-thalassemia diagnosis.

Globin chain synthesis was studied in 13 iron-deficient patients. The mean whole-cell globin alpha/beta ratio in the peripheral blood of 11 patients was 1.05 +/- 0.06 which is similar to the value 0.99 +/- 0.08 obtained for 10 controls. The ratios obtained for stroma-free globin were not significantly different from those of whole cell preparations. In contrast, the alpha/beta ratio of bone marrow was 0.73 +/- 0.14 in 10 iron deficient patients, which is significantly lower than that of controls. Two other patients had decreased alpha/beta ratios in the peripheral blood, probably because of the presence of an alpha-thalassemia gene. These results demonstrate a reduced rate of synthesis of alpha chains relative to that of beta chains in the bone marrow of iron-deficient patients that is not demonstrable in the peripheral blood.

Globin chain synthesis has been studied in 17 patients with thalassemia intermedia, and their relatives, also investigated by routine hematologic and hemoglobinic tests. The mean α/non α ratio was alw

By hybridization with [3H]labeled globin cDNA the contents of globin coding sequences in total nuclear RNA, poly(A)+nuclear RNA, poly(A)--nuclear RNA and polysomal RNA of chicken immature red blood cells was determined to be 0.86%, 20%, 0.42% and 1% respectively. As the poly(A)+-fraction comprises only about 2% of total nuclear RNA, globin coding sequences are distributed with 49% in the poly(A)+-fraction and with 51% in the poly(A)--fraction.

Studies were carried out during the 1st yr of life in normal infants born at term to determine the proportions of fetal hemoglobin (Hb F) and adult hemoglobin (Hb A) being synthesized, in order to describe the complete switchover from Hb F to Hb A synthesis during postnatal life. 53 blood samples from 37 infants were incubated in an amino acid mixture containing [14C]leucine and chromatographed on DEAE-Sephadex for separation of Hb F and Hb A fractions. The completeness of the CEAE-Sephadex separation of Hb A and Hb F at an age when the major portion of synthesis was of the adult type of hemoglobin was confirmed by globin chain chromatography with the use of carboxylmethyl cellulose. There was a rapid decline in Hb F synthesis postnatally until 16-20 wk of age when levels of 3.2% plus or minus SD 2.1% were reached. By combining this data with that previously published, the complete switchover from Hb F to Hb A synthesis can be described in humans in relation to postconceptional age. It follows a sigmoid curve; the steep portion, which lies between the 30th and 52nd postconceptional week, is preceded and follwoed by plateaus averaging 95% and 7% Hb F synthesis, respectively.

The question, “Is Hb Gα Philadelphia linked to α-thalassaemia?” was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 α-chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had β+ thalassaemia and the other was heterozygous for the Gα Philadelphia chain gene. The former had a globin chain production ratio α/β well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the β+ thallassaemia from one parent and the Gα Philadelphia chain gene from the other and showed the typical picture of α/β-thalassaemia (α/β ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 α-chain genes.