A B S T R A CT 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 Ay-or fl-globin synthesis was determined from the total of y + P, and the percent Gy from the total of Gy + Ay. The nonparametric correlation coefficients of percent G-y with percent y or # were obtained. Each group of colonies at each time point was examined separately. In colonies from adult blood, the proportion of Gy-synthesis did not correlate with the proportion of y-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 Gy with f3-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 Gy with A-synthesis. These findings are compatible with a clonal model for hemoglobin switching. Fetal progenitors, in which G'y-and #-syntheses are negatively correlated, are gradually replaced during ontogeny by adult progenitors. The adult progenitors produce more If (less y), and the proportions of Goy-and y-or Bi-synthesis are not correlated.

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.

Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.