A B S T R A C T Hereditary pyropoikilocytosis (HPP)is a hemolytic anemia characterized by microspherocytosis, poikilocytosis, and an unusual thermal sensitivity of erythrocytes. We have investigated the contribution of abnormal membrane skeletal assembly to these abnormal HPP erythrocyte properties. Skeletons prepared from fresh HPP ghosts with Triton X-100 were considerably more fragile than skeletons from control erythrocytes. Spectrin, the major skeleton component, extracted at 0°C from normal erythrocytes, was present primarily as tetramers and high molecular weight complexes. In contrast, spectrin extracted from HPP erythrocytes under identical conditions contained a significant amount of dimers with a concomitant decrease of tetramers. Furthermore, spectrin dimers from HPP erythrocytes differed from normal spectrin dimers in their failure to reassociate into tetramers both in solution and in the membrane. Presumptive HPP carriers (asymptomatic mothers of the two patients) exhibited a mild but reproducible increase of spectrin dimers in 0°C extracts and a defective reassociation of spectrin dimers of tetramers both in solution and in the membrane. We conclude that in HPP, self-association of spectrin dimers into tetramers is defective, which accounts for the instability of membrane skeletons.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is probably the most common disease-producing genetic polymorphism of humans. Virtually all G6PD-deficient Africans show the G6PD A- phenotype, an electrophoretically rapid, deficient enzyme. The recently acquired ability to identify the point mutations producing the different variants has given us new insights into the population genetics of G6PD variants. Twenty-nine males with the G6PD A- phenotype were studied. They were of African, Mexican, Spanish, and US white ethnic origin. All had the A---G transition at nucleotide 376 characteristic of G6PD A. In each case, one of three additional mutations was present, at nucleotides 202, 680, or 968. That in this population second mutations producing G6PD deficiency occurred only on the genetic background of G6PD A suggests that G6PD A was at one time the most common type of G6PD in Africa. However, the nucleotide sequence of the chimpanzee (Pan troglodytes) G6PD indicates that the primordial human type of G6PD was G6PD B.

We examined erythrocytes from 18 patients with hereditary elliptocytosis. Spectrin from eight patients (referred to as type 1) was defective in dimer-dimer association as demonstrated in two ways. First, there was an increased amount of spectrin dimer with a concomitant decrease in tetramer as measured in erythrocyte membrane preparations extracted at D.C under low-salt conditions (the amount of spectrin dimer was 15-33% of total spectrin species compared with a normal range of 3-7%). Second, the equilibrium constants of spectrin dimerdimer association were decreased in both solution and in situ membrane. Spectrin from the remaining 10 patients (referred to as type 2) showed normal dimer-dimer association. Membrane skeletons, produced from ghosts of both types of hereditary elliptocytosis by Triton X-100 extraction, were unstable when mechanically shaken. Because spectrin tetramers, but not dimers, can crosslink actin, we postulate that the defective spectrin dimer-dimer association in type 1 diminishes actin crosslinking and thus is responsible for membrane skeletal instability. A defective protein-protein association in type 2, however, remains to be identified. Spectrin, together with actin, band 4.1, and some minor components, forms a two-dimensional submembrane skeletal network that is thought to stabilize the membrane structure and maintain the discoid shape ofnormal erythrocytes (for reviews, see refs. 1 and 2). Recent studies suggest that membrane skeletal defects are involved in several erythrocyte disorders, including hereditary spherocytosis, pyropoikilocytosis, and elliptocytosis (2-5). In patients who have hereditary elliptocytosis (HE), it has been found that membrane skeletons derived from HE erythrocytes retain their abnormal elliptic shape and that purified spectrin from some, but not all, is abnormally heat sensitive (2). However, the pathogenesis of this disease is not understood. In a preliminary examination, we have observed a decreased stability of HE membrane skeletons on mechanical shaking. In a subpopulation of HE patients, this was associated with a concomitant decrease in spectrin tetramers and increase in spectrin dimers (6). We-now demonstrate in these patients a defective association of spectrin dimers into tetramers in both the membrane and solution and point out the heterogeneity of such a defect among unrelated HE patients.EXPERIMENTAL PROCEDURES Clinical Material. We have studied 18 HE patients from 13 unrelated families. Blood films from all subjects showed 60% or more elliptical erythrocytes. Clinical review suggested minimal or absent hemolysis. Patients were 1.5-82 years old.Venous blood from normal and HE-patients was collected in sterile tubes containing citrate/phosphate/dextrose or citrate/ dextrose. Specimens that were not obtained at St. Elizabeth's Hospital, Boston, were transported in insulated containers with ice to Boston where they were kept at %40C and analyzed no more than 3 days later. Spectrin Extraction. Ghosts were prepared from washed erythrocytes ...

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