Studies of hemoglobin expression in erythroid cells of early human fetuses using anti-gamma- and anti-beta-globin chain fluorescent antibodies.

Cells from early human fetuses were analyzed for gamma- and beta-globin expression by means of monospecific and monoclonal anti-gamma- and anti-beta-chain antibodies. We found that gamma-chain synthesis in embryonic (yolk sac) nucleated cells is either absent or it is below the threshold of sensitivity of the fluorescent antibody method (about 0.1 pg of hemoglobin per cell or less).

Hemoglobin switching activity.

We describe observations that suggest the existence of an activity that induces the forward HbF-to-HbA switch. Studies in normal adult BFU-E cultures, in cultures of mutant BFU-E, and in cultures of cells from the neonatal and fetal stages of development reveal that the cells from various stages of ontogeny respond differently to this activity. Experiments using transfers of erythroid clones indicate that the hemoglobin switching activity acts on progenitors and that the interaction is of a direct nature.

Hemoglobin switching in culture: evidence for a humoral factor that induces switching in adult and neonatal but not fetal erythroid cells.

An erythropoietic activity that exerts a profound effect on fetal Hb synthesis is present in fetal sheep sera and it attains a peak concentration at the end of the second to the middle of the third trimester of fetal life. The activity consistently inhibits the increased synthesis of fetal Hb in cultures of burst-forming units (BFUes) from normal adults. In cultures of BFUes from homozygous beta+-thalassemias the activity produces a striking decline in gamma chain synthesis, a decline in G gamma/A gamma chain synthesis ratio, and an increase in delta/gamma and alpha/non-alpha ratios--i.

Alpha-thalassemia in two Mediterranean populations.

We used restriction endonuclease analysis to determine the incidence of alpha-thalassemia in two Mediterranean islands. In a random population sample, the gene frequency of deletion-type alpha-thalassemia-2 (-alpha) was 0.18 in Sardinians and 0.

Greek (A gamma) variant of hereditary persistence of fetal haemoglobin: globin gene organization and studies of expression of fetal haemoglobins in clonal erythroid cultures.

Individuals heterozygous for the Greek (A gamma) variant of hereditary persistence of fetal haemoglobin (HPFH) synthesize Hb F whose gamma-globin chains are predominantly of the A gamma type. DNA obtained from Greek HPFH heterozygotes was used to test for abnormalities in the organization of non alpha-globin genes. In addition, gamma- and beta-globin expression was studied in BFUe cultures.

De novo mutations producing unstable Hbs or Hbs M. II. Direct estimates of minimum nucleotide mutation rates in man.

Cases of unstable hemoglobin and hemoglobin M disease that have appeared as de novo mutants over a span of approximately 50 years were used to deriving minimal, direct estimates of mutation rates per nucleotide per generation in man. The estimates are based upon analysis of data related to 40 cases of unstable Hbs and 15 of Hbs M that arose in 13 countries. The estimated rate calculated using all de novo beta-gene mutants is 7.

Human hemoglobin switching: insights from studies of erythroid cultures.

Clonal erythroid cultures have been used in the investigation of the cellular mechanisms underlying the switch from fetal to adult Hb formation during ontogeny and the expression of fetal hemoglobin in adult life. It has been shown that adult BFUes have the potential to form F cells and A cells in culture. The cellular segregation in the expression of Hb F within a clone leads to the formation of characteristic bursts displaying a bimorphic F+/F-pattern of Hb F expression.

Globin synthesis in erythroid bursts that mature sequentially in culture. I. Studies in cultures of adult peripheral blood BFU-Es.

To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU-Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week.

Stochastic expression of fetal hemoglobin in adult erythroid cells.

The expression of fetal hemoglobin, Hb F, in the adult cells is cellularly restricted both in vivo and in culture. Because, in cultures of erythroid progenitors, subclones that express or fail to express Hb F are derived from the same erythroid stem cell, a mechanism must exist whereby Hb F expression segregates in the progeny of erythroid progenitors during their differentiation. We present mathematical analyses of experimental data which suggest that expression of Hb F in human adult erythroid cells occurs on a stochastic basis.

Mapping of antigenic sites on human haemoglobin by means of monoclonal antibodies and haemoglobin variants.

Monoclonal antibodies specific for human globin chains have been prepared and the following strategy has been applied in delimiting the antigenic sites involved in antibody binding. The structural sites of the human globin subunit that might be recognised by the monoclonal antibody were deduced from comparisons of the primary structures of mammalian globin chains that did or did not react with the antibody. The involvement of individual residues at these specific sites was subsequently tested by reacting the antibody with abnormal human haemoglobins in which there was either a substitution or a deletion of one of the residues in question.

De novo mutations producing unstable hemoglobins or hemoglobins M : I. Establishment of a Depository and use of data to test for an association of de novo mutation with advanced parental age.

Hemoglobins M and unstable hemoglobins cause clinical syndromes that are transmitted in autosomal dominant fashion. Pedigrees of 50 probands with de novo mutations producing unstable Hb disease or Hb M disease were compiled. Cases were ascertained (1) by screening the relevant literature published from 1950 through 1980 and (2) through personal communication.

Cell sorter immunofluorescence detection of human erythrocytes labelled in suspension with antibodies specific for hemoglobin S and C.

We have developed an immunochemical method for labeling human red blood cells in suspension with hemoglobin-specific antibodies. A membrane permeable cross-linking reagent, dimethyl suberimidate, is used to covalently bind, in situ, a fraction of the intracellular hemoglobin to integral membrane proteins. Hypotonic lysis and washing of the cells removes the unbound hemoglobin resulting in red blood cell ghosts which are permeable to macromolecules.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin hasharon, a human alpha-chain variant.

Antibodies against the abnormal human hemoglobin, Hb Hasharon (alpha 47 Asp leads to His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.

Two siblings with unusually mild homozygous beta-thalassemia: a didactic example of the effect of a nonallelic modifier gene of the expressivity of a monogenic disorder.

Two adult Black sibs with homozygous beta-thalassemia had severe deficiency of beta-chain production but an unusually mild clinical course and almost normal hematocrit values. Their father had the typical hematological findings of beta-thalassemia trait but the mother, in addition to the beta-thalassemia trait, had elevated F cells (42.2%).

The switching from hemoglobin F to hemoglobin A formation in man: parallels between the observations in vivo and the findings in erythroid cultures.

The findings from studying Hb F synthesis in BFUe cultures parallel the findings in vivo. The BFUe's which are explanted from fetuses and neonates and seeded in culture behave as if they are already preprogrammed, in vivo, to follow a defined behavior in culture and produce erythroblasts with programs very similar to those of the cells in vivo. In the adult, the discrepancy between the low in vivo Hb F expression and the enhanced Hb F synthesis in culture can be explained on the same basis by which the appearance of F cells in vivo is explained: the normal in vivo environment is such that few cells take the F cell path; in vitro the environmental conditions are such as to induce formation of many F+ committed progeny from BFUe's.

Development of a somatic mutation screening system using Hb mutants. IV. Successful detection of red cells containing the human frameshift mutants Hb Wayne and Hb Cranston using monospecific fluorescent antibodies.

The production and purification of antibodies detecting Hb Wayne, an alpha-globin frameshift mutant, and Hb Cranston, a beta-globin frameshift mutant, are described. The antibodies are of a nonprecipitating nature, and they permit strong fluorescent labeling of erythrocytes containing Hb Wayne or Hb Cranston. Studies using artificial mixtures containing cells with either of the two mutants in frequencies ranging from 1 in 10(2) to 1 in 10(5) showed that fluorescent antibodies can detect rare mutant red cells in the presence of vast excesses of normal erythrocytes.

Fetal Hb production during acute erythroid expansion. I. Observations in patients with transient erythroblastopenia and post-phlebotomy.

In order to study fetal haemoglobin production during acute erythroid expansion we did sequential measurements of Hb F-containing erythrocytes (F-cells) and of relevant haematological parameters in 10 subjects recovering from eyrthroid aplasia, iron deficiency anaemia or following phlebotomy. An increased production of F-cells was consistently observed during the acute marrow expansion, but there were significant differences in the maximum F-cell response among individuals. These differences could not be explained by differences in the degree of anaemia alone, nor could they be correlated with the level of peak reticulocytosis.

Triplicated alpha-globin loci in humans.

We have identified 12 individuals who are heterozygous for a chromosome with three alpha-globin genes. We determined the presence of the third alpha-globin locus by restriction endonuclease digestion and hybridization with alpha-globin cDNA probes. The three alpha-globin loci resided in an elongated fragment on digestion with EcoRI, BamHI, and Xba I, and the third locus was present in an additional 3.