Blanche P. Alter

Globin Chain Electrophoresis: a New Approach to the Determination of the Gγ/Aγ Ratio in Fetal Haemoglobin and to Studies of Globin Synthesis

Summary. Separation of globin chains by electrophoresis provides a simple and rapid method for the determination of the Gγ/Aγ ratio in human fetal haemoglobin, and of biosynthetic rates of the globin chains. Whole haemolysates were analysed by electrophoresis on polyacrylamide gels in urea, acetic acid and Triton X‐100. Electrophoresis of haemolysates from newborn infants led to four bands: Aγ, Gγ, β and α. The identity of these bands was indicated by examination of haemoglobins of known globin chain composition. In 15 samples, the %Gγ was similar by Triton gels and by amino acid analysis of the γCB‐3 peptide. Some mutant globin chains were also separable with the electrophoretic technique. Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.

Application of endonuclease mapping to the analysis and prenatal diagnosis of thalassemias caused by globin-gene deletion.

We applied a recently developed and more direct technic to diagnose thalassemia syndromes associated with deletion of particular globin structural genes and to assess a fetus at risk for one of those conditions, deltabeta-thalassemia. The method allows assessment of the globin genes present in total cellular DNA and is applicable to amniotic-fluid cell DNA. Cellular DNA fragments produced by cleavage using two specific restriction endonucleases are separated on the basis of size by agarose-gel electrophoresis, and the distribution of specific sequences among the DNA fragments determined by molecular hybridization. We observed the total deletion of alpha-globin genes in homozygous alpha-thalassemia (hydrops fetalis with hemoglobin Barts) and the deletion of particular beta and beta-like sequences in cases homozygous for hereditary persistence of fetal hemoglobin and deltabeta-thalassemia. Analysis of amniotic-fluid cell DNA from a fetus at risk for deltabeta-thalassemia demonstrated the feasibility of these improved methods for antenatal diagnosis. The molecular studies confirmed the diagnosis predicted by analysis of fetal blood and established at birth.

Prenatal diagnosis of hemoglobinopathies. A review of 15 cases.

We attempted prenatal diagnosis of hemoglobinopathies in 15 cases--11 for beta-thalassemia and four for sickle-cell disease. Fetoscopy was used in seven cases, and placental aspiration in eight. One premature labor, with fetal loss, followed placental aspiration. Globin synthesis was assessed by incubation of samples with 3H-leucine and chain separation on carboxymethylcellulose columns. Homozygous disease was predicted in two pregnancies, which were interrupted, and the diagnosis confirmed. In one case homozygosity was suspected. A repeat test was advised but not accepted. The fetus had thalassemia trait. One pregnancy was interrupted despite our prediction of thalassemia trait. Eight pregnancies went to term. Seven predictions that the infants would not have homozygous disease were confirmed. One prediction of sickle trait proved to be sickle-cell disease. Although prenatal diagnosis of hemoglobinopathies is feasible, the present frequency of fetal loss and diagnostic error indicates need for improvement.

Switch from fetal to adult hemoglobin is associated with a change in progenitor cell population.

To examine the switch from fetal to adult hemoglobin at the cellular level, erythroid progenitor cells from newborn infants and adults were cultured in methyl cellulose with erythropoietin. Individual erythroid colonies were labeled with [3H]leucine at various times, and globin synthesis patterns examined by gel electrophoresis and fluorography. The percent gamma- or beta-globin synthesis was determined from the total of gamma + beta, and the percent G gamma from the total of G gamma + A gamma. The nonparametric correlation coefficients of percent G gamma with percent gamma or beta were obtained. Each group of colonies at each time point was examined separately. In colonies from adult blood, the proportion of G gamma-synthesis did not correlate with the proportion of gamma-synthesis. Colonies from newborn blood fell into two groups. Those that developed from relatively mature progenitor cells, and were seen on day 14, showed a strong negative correlation of G gamma with beta-globin synthesis. However, those newborn colonies that developed from immature progenitors, and were seen later in culture (days 17 and 21), showed no correlation of G gamma with beta-synthesis. These findings are compatible with a clonal model for hemoglobin switching. Fetal progenitors, in which G gamma- and beta-syntheses are negatively correlated, are gradually replaced during ontogeny by adult progenitors. The adult progenitors produce more beta (less gamma), and the proportions of G gamma- and gamma- or beta-synthesis are not correlated.

Prenatal diagnosis of hemoglobin opathies and other hematologic diseases

P R E N A T A L D I A G N O S I S of hemoglobinopathies and selected other hematologic diseases is now possible due to recent developments in molecular biology and biochemistry, combined with techniques for obtaining samples of fetal amniotic fibroblasts or fetal blood. Most of the groundwork and most of the cases have involved the hemoglobinopathies (fl- and a-thalassemia and sickle cell disorders), but the methods and approaches will have more general application. This review is focused on the hemoglobin disorders, but other blood diseases are discussed briefly. BACKGROUND Detection of hemoglobinopathies is predicated on an understanding of the molecular aspects of hemoglobin production, which are outlined in Fig. 1. Globin genes in DNA are transcribed into heterogeneous high molecular weight RNA, which is further processed to produce small.

Deletion of the A gamma-globin gene in G gamma-delta beta-thalassemia.

In an individual homozygous for G gamma-delta beta-thalassemia, a physical alteration in gamma-globin gene organization was detected by restriction enzyme mapping. The data indicated that the absence of A gamma-globin chains resulted from extension of the DNA deletion from the delta beta-globin gene region into the gamma-globin gene region rather than a functional disturbance of gamma-gene expression.

PRENATAL DIAGNOSIS OF HEMOGLOBINOPATHIES: THE NEW ENGLAND APPROACH*

Prenatal diagnosis of hemoglobinopathies was first attempted in 1974. Since then, more than 500 cases have been studied in approximately a dozen centers worldwide. The method that is employed most frequently involves sampling of fetal blood in utero by fetoscopy or placental aspiration at 18 to 20 weeks gestation. Globin chain synthesis is then examined by column chromatography. The application of these techniques is reviewed in the articles by Kan and Alter and their colleague^.-^ In specific cases in which the globin genes are absent or altered, fetal amniotic fibroblast DNA can be used to provide diagnostic information by liquid hybridization (absent genes) or restriction enzyme map techniques (absent or altered genes). These conditions include a, @?, and rare types of /3O thalas~emia ,~‘ and some cases of sickle-cell disease.8 However, most cases of /3” and all /3+ thalassemias have normal gene maps. Thus, study of globin chain syn-

Beta-thalassaemia trait: imprecision of diagnosis at birth.

Globin chain synthesis was studied at birth in infants who had been tested for haemoglobinopathies while in utero. Subsequent haematological studies showed that five infants were normal, five had β‐thalassaemia trait, and two had α‐thalassaemia trait. Correct assignment of these diagnoses could not be made in individual cases from globin chain studies at birth, although the β/α ratios did cluster into groups. By total radioactivity, the mean β/α ratio was 0.38 in the normal infants, and 0.22 in those with β‐thalassaemia trait. Mean (β+γ)/α was 0.90 in the former, and 0.70 in the latter. Specific activity ratios (counts per minute, per absorbance at 280 nm) failed to distinguish thalassaemia trait from normal, as a group or individually. Accurate diagnosis of β‐thalassaemia trait required classical haematological studies during the first year of life.

Prenatal Diagnosis of Sickle-Cell Anemia and Alpha G-Philadelphia: Study of a Fetus Also at Risk for Hb S/β+- Thalassemia

Prenatal diagnosis is now available as a research procedure for families whose children are at risk for hemoglobinopathies. The expressions of the sickle1 2 3 and thalassemia genes4 , 5 have been d...

F-cell regulation.

Fetal hemoglobin production can ameliorate Cooley’s anemia in part because gamma chains solubilize excess alpha chain and thereby reduce erythroid cell destruction.’ Since fetal hemoglobin is regularly observed in the blood of patients with Cooley’s anemia, effective pharmacologic manipulation of its production has been long sought. Encouragement has been gained from studies in which fetal hemoglobin synthesis can be demonstrated in cultures of colonies derived from human hematopoietic progenitors. In fact, some experiments suggest that erythropoietin itself may stimulate human gamma chain synthesis in vitro and certain primates exhibit a prompt increase in fetal hemoglobin synthesis in response to hypoxia and hemolysis.4 But enthusiasm is tempered because the increased fetal hernoglobin seen in nondeletion forms of Cooley’s anemia is due mainly to the production of F cells, a common occurrence in many forms of accelerated erythropoiesis,5 and to their selective intramedullary and intravascular survival in Cooley’s anemia.6 It should be emphasized that F cells differ markedly from fetal cells in that their average fetal hemoglobin content is about 15% of the total intracellular hemoglobin. They should perhaps more properly be called FA cells. Since increased production of F cells would be of considerable therapeutic benefit in Cooley’s anemia, clinical and experimental studies of F-cell regulation are of potential importance. The purpose of this paper is to present a brief review of some other clinical circumstances in which accelerated F-cell production is observed, to review our experience with F-cell production in vitro, and finally to propose a model of erythropoiesis that may explain, at least in part, our in vivo and in vitro observations.