Normal Spectrin Research Articles

Limb-girdle weakness in a marfanoid man: distinguishing calpainopathy from Becker's muscular dystrophy

A 24-year-old man had experienced hip-girdle weakness since the age of 15 years and exercise-induced muscle cramps with ‘toe-walking’ since aged 12 years. He was thought to have Marfan's syndrome...

An unexpectedly bizarre blood film in hemoglobin H disease

A 3-year-old boy from Saudi Arabia had been followed up with a diagnosis of hemoglobin H disease, identified in a neonatal screening programme. His parents were second cousins. Alpha globin genotyping at birth had revealed homozygosity for the non-deletional Saudi-T mutation (αTα/αTα), both parents being carriers. He had remained clinically asymptomatic. Red cell indices at the age of three years were: RBC 3.84 x 1012/l, hemoglobin concentration 80 g/l, MCV 61 fl, MCH 20.9 pg, MCHC 34 g/l, red cell distribution width 34.8 (normal range 11.8-14.3) and reticulocyte count 447 x109/l. His blood film, in addition to microcytosis, showed gross poikilocytosis with the abnormal red cells including elliptocytes and many bizarre poikilocytes, some very small (image). Although poikilocytosis can be prominent in hemoglobin H disease, we have not seen a case with this degree of abnormality. The film was reminiscent of hereditary pyropoikilocytosis. For this reason red cell membrane studies were initiated. An eosin-5 maleimide (EMA)-binding assay [1] gave a twin peak fluorescence profile: 57% of phenotypically normal red cells (MCF = 64.7 units) and 42% having reduced fluorescence (MCF = 32.4 units). Spectrin (Sp) analysis showed a Sp dimer content of 25.6 %, compared with 7.4 % for a normal control within the same assay. In limited trypsin digestion of spectrin, SpαI/50 variant was detected in the child, and absent in the mother. Neither had an increase of SpαV/41 tryptic peptide, thus excluding the presence of the polymorphic low expression allele, SpαLELY. Maternal red cells were not elliptocytic and had normal spectrin dimer content and normal EMA binding. A paternal sample was not available for testing. Although the EMA-binding assay has high specificity and sensitivity for hereditary spherocytosis, it can also be abnormal in hereditary pyropoikilocytosis, Southeast Asian ovalocytosis, cryohydrocytosis, and some cases of congenital dyserythropoietic anemia type II [2]. Hereditary elliptocytosis usually gives a single fluorescence peak within the range for normal red cells but occasionally twin peaks are observed. In this child with co-inheritance of hemoglobin H disease and hereditary elliptocytosis, the severity of the hemolytic anaemia is that expected in uncomplicated hemoglobin H disease but the bizarre blood film reflects an interaction of the hemoglobinopathy and the membrane defect. 1

αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

Novel Exon 2 Alpha Spectrin Mutation in a Caucasian Family with Pyropoikilocytosis, Elliptocytosis, and Normal Morphology: Three Phenotypes Due to Decreased Spectrin mRNA and Intragenic Crossover

Mutations of alpha spectrin involving the heterodimer self-association site represent a common group of membrane skeletal defects resulting in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, hemolytic anemia, severe spectrin dimer self-association defect, and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for a functional alpha spectrin defect that interferes with tetramer assembly due to missense mutations (commonly present in exon 2), and a second defect that results in decreased synthesis of normal alpha spectrin. In contrast, HE subjects have normal spectrin content and a less severe self-association defect. The modifying factor accounting for decreased synthesis of alpha spectrin is a hypomorphic mutation termed alpha spectrin LELY. LELY is a low-expression allele of the alpha spectrin gene characterized by a C>G mutation in codon 1857 of exon 40 and a C>T mutation in intron 45 resulting in decreased assembly of alpha/beta spectrin dimers. Here, we describe a family of Northern European descent in which six members in three generations were heterozygous for a novel mutation in alpha spectrin exon 2, codon 34: CGG->CCG (Arg>Pro) and for alpha spectrin LELY inherited in trans. Affected members harboring this novel exon 2 mutation exhibit three different RBC phenotypes: atypical HPP (1/6), HE (4/6), and normal (1/6) (see figure). Quantitation of RBC membrane proteins in subjects with abnormal RBC morphology revealed an increase in spectrin dimers due to an increase in the mutant 74kD alpha spectrin peptide. There was a significant association of abnormal morphology with the levels of dimers and mutant 74kD peptide (N<HE<HPP). In addition, HPP and HE subjects had a greater expression of mutant exon 2 allele compared to wild type (by allele specific PCR) at a ratio approximating 3:1. Moreover, the HPP subject had a decreased amount of reticulocyte total alpha spectrin mRNA (~10% of normal) while HE individuals had only a slight decrease in reticulocyte total alpha spectrin mRNA (~ 85% of normal). One individual (II-6), who had an exon 2 missense mutation and homozygous intron 45 LELY mutation exhibiting normal RBC morphology, was investigated in greater detail. After ruling out non-paternity by standard testing, genotyping of his alpha spectrin gene was performed and an intragenic crossover detected, resulting in homozygosity for the intron 45 LELY mutation and wild type exon 40 (see figure). We conclude that the phenotype of individual II-6 is ameliorated by this de novo mitotic event. The HPP phenotype of the propositus is likely due to decreased transcription of both alpha spectrin alleles; however, its molecular basis remains to be determined. # Equal contribution. [Display omitted]

A Novel Mutation of the α Spectrin Gene in a Family of Northern European Descent Is Associated with Three Different Phenotypes.

Mutations of α spectrin (Sp) involving the Sp heterodimer self-association site (the α I domain of Sp) represent the most common group of membrane skeletal defects in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, a severe spectrin dimer self-association defect and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for an α sp defect that interferes with sp tetramer assembly and a second defect which results in decreased synthesis of functional α sp. In contrast, HE subjects have normal spectrin content and a less severe sp self-association defect. The clinical expression of HE/HPP is influenced by the inheritance of modifying factors such as the α Sp hypomorphic mutation, α LELY. α LELY is a low expression allele of the α Sp gene characterized by a C>G mutation in codon 1857 of exon 40 and a C>T-12 mutation in intron 45 that is responsible for partial skipping of exon 46, which is essential for the functional assembly of α/b sp dimers. Here we describe a family of northern European descent in which members had different erythrocyte morphology ranging from atypical HPP to HE to normal and a novel αSp mutation. Quantitation of RBC membrane proteins of the propositus with atypical non-microcytic HPP revealed 48% spectrin dimers (control 10%) due to a marked increase in the 74kD αI Sp peptide. There was only a slight decrease in the spectrin/band 3 ratio, which correlated with the normocytic morphology. There was also an abnormal α Sp peptide at 41kD suggesting presence of α LELY. Sequencing of his α Sp gene revealed heterozygosity for a novel mutation in exon 2, codon 34: CGG->CCG (Arg>Pro) and heterozygosity for α LELY. These mutations were also present in his brother and daughter who have HE, while another son, who had Arg34Pro, but not α LELY has repeatedly confirmed normal morphology (data in pedigree, figure 1). Ongoing clinical, α Sp peptide, DNA and quantitative Real Time PCR mRNA studies of extended family reveal a complex interaction of codon 34: G>C mutation with α LELY and different levels of transcripts of α Sp alleles. #equal contribution. [Display omitted]

Pathogenic proline mutation in the linker between spectrin repeats: disease caused by spectrin unfolding

AbstractPathogenic mutations in α and β spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin α-βtetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of α-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5. Results show that the linker mutation destabilizes a significant fraction of the 5-repeat construct at 37°C, whereas the WT remains fully folded well above body temperature. In WT protein, helical linkers propagate stability from one repeat to the next, but the mutation disrupts the stabilizing influence of adjacent repeats. The results suggest a molecular mechanism for the high frequency of disease caused by proline mutations in spectrin linkers.

Membrane Perturbations of Erythrocyte Ghosts by Spectrin Release

The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.

Recurrent fatal hydrops fetalis associated with a nucleotide substitution in the erythrocyte beta-spectrin gene.

We studied a kindred in which four third-trimester fetal losses occurred, associated with severe Coombs-negative hemolytic anemia and hydrops fetalis. Postmortem examination of two infants revealed extensive extramedullary erythropoiesis. Studies of erythrocytes and erythrocyte membranes from the parents revealed abnormal erythrocyte membrane mechanical stability as well as structural and functional abnormalities in spectrin, the principal structural protein of the erythrocyte membrane. Genetic studies identified a point mutation of the beta-spectrin gene, S2019P, in a region of beta spectrin that is critical for normal spectrin function. Both parents and two living children were heterozygous for this mutation; three infants dying of hydrops fetalis were homozygous for this mutation. In an in vitro assay using recombinant peptides, the mutant beta-spectrin peptide demonstrated a significant abnormality in its ability to interact with alpha spectrin. This is the first description of a molecular defect of the erythrocyte membrane associated with hydrops fetalis.

Gaps in the erythrocyte membrane skeleton: A stretched net model

The geometry of spectrin-free regions in the erythrocyte membrane skeleton is modeled using Monte Carlo calculations for an incomplete triangular lattice of entropy springs under tension. Intact springs correspond to normal spectrin molecules, and cut springs correspond to spectrin that is missing or unable to associate normally. As springs are cut and the network is allowed to relax to mechanical equilibrium, gaps in the network appear. Geometrical properties of these gaps are obtained as a function of the fraction of springs cut. The most important property modeled is the area of the largest spectrin-free region; this area increases approximately exponentially as the fraction of normal spectrin decreases from 100% to approximately 50%. The effect of these gaps on lateral diffusion and vesiculation is discussed.

Oxidation of Spectrin and Deformability Defects in Diabetic Erythrocytes

We reasoned that de novo oxidative damage, as a result of increased protein glycosylation, could participate in the mechanisms whereby diabetic erythrocytes acquire membrane abnormalities. To examine this hypothesis, the extent of erythrocyte membrane protein glycosylation and the oxidative status of spectrin, the major component of the erythrocyte membrane skeleton, were examined. Labeling erythrocyte membranes with [3H]borohydride, which labels glucose residues bound to proteins, revealed that several proteins were heavily glycosylated compared with nondiabetic erythrocyte membranes. In particular, the proteins beta-spectrin, ankyrin, and protein 4.2 were the most glycosylated. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis of diabetic erythrocyte membranes did not reveal any quantitative or qualitative abnormalities in spectrin or other membrane proteins, examination of spectrin oxidative status by amino acid analysis and with cis-dichlorodiammineplatinum(II) (cDDP), a chemical probe specific for protein methionine and cysteine residues, demonstrated that the diabetic spectrin was oxidatively damaged: spectrin from diabetic subjects contained 35% less methionine (P less than 0.002), 15% less histidine (P less than 0.006), and a twofold increase in cysteic acid (P less than 0.001) compared with normal spectrin. Diabetic spectrin bound 32% less cDDP than normal spectrin (P less than 0.001); the lowest cDDP binding was observed with spectrin from insulin-dependent diabetic subjects. The extent of cDDP binding to diabetic spectrin correlated moderately and inversely with glycosylated hemoglobin (GHb) levels (n = 12, r = -0.727). Erythrocyte deformability, measured by ektacytometry, was decreased between 5 and 23% of control measurements (average of approximately 10%) in 21 of 32 diabetic subjects surveyed.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular defect of truncated beta-spectrin associated with hereditary elliptocytosis. Beta-spectrin Gottingen

A large German family with autosomal dominantly inherited elliptocytosis, associated with truncated beta-spectrin missing the phosphorylated C-terminal peptide, has been described (Eber, S.W., Morris, S. A., Schroeter, W., and Gratzer, W. B. (1988) J. Clin. Invest. 81, 523-530). We have attempted to delineate the molecular defect of this abnormality at the gene level. Southern blot analyses revealed no evidence of a partial gene deletion or rearrangement. We used polymerase chain reaction (PCR) and amplified several relevant portions of the beta-spectrin gene, using genomic DNA from two different heterozygous patients. No abnormality was found in the last four exons of the beta-spectrin gene produced by PCR. Examination of the introns connecting the last four exons revealed a T to A substitution in the 5' splice site following the exon X in four of eight clones prepared from two affected individuals, but not from a normal subject of this family. To examine the effect of the T to A substitution in these patients, we made cDNA from the reticulocyte mRNA of the patient and examined its composition by PCR. Two distinct PCR fragments produced from the patient's beta-spectrin cDNA were found. One matched the predicted size for normal spectrin, while the other was 197 base pairs shorter. The mutant cDNA sequence revealed that the entire exon preceding the T to A substitution was spliced out, while the two terminal exons were preserved. The deletion of this exon resulted in a frameshift giving a different terminal amino acid (serine instead of leucine) as well as a new termination codon causing a deletion of 129 amino acids including potentially phosphorylated residues.

Variable clinical severity of hereditary spherocytosis: Relation to erythrocytic spectrin concentration, osmotic fragility, and autohemolysis

To determine whether stratifying hereditary spherocytosis by degree of severity could provide guidelines regarding which patients would benefit from splenectomy, we evaluated the clinical characteristics of 80 patients (63 children) and 27 healthy relatives. In addition to routine hematologic determinations, osmotic fragility, autohemolysis, erythrocyte spectrin content, and erythrocyte membrane lipid phosphorus were measured and correlated with the disease severity. Four categories were identified: (1) spherocytosis as a trait in symptom-free relatives of patients with recessively inherited disease; (2) mild and (3) moderate spherocytosis, largely observed in patients with dominantly inherited disease; and (4) severe spherocytosis, observed in only two patients, who were characterized by recessive inheritance and transfusion dependence. By the identification of carriers, a recessive mode of inheritance could be demonstrated in 20% of the families with spherocytosis. The erythrocyte spectrin concentration was normal in carriers and patients with mild spherocytosis, and was significantly reduced in the moderate and severe states of the disease. This difference was not accounted for by reduced membrane area of the cells, as measured by the phospholipid concentration per cell. We conclude that patients with mild spherocytosis usually do not require splenectomy during childhood and adolescence; patients with moderate or severe disease should have splenectomy. Patients with severe spherocytosis have a partial response to splenectomy but a considerable degree of increased hemolysis persists. Most patients with less than 80% of normal spectrin content require splenectomy.

Alteration of the Erythrocyte Membrane Skeletal Ultrastructure in Hereditary Spherocytosis, Hereditary Elliptocytosis, and Pyropoikilocytosis

Abstract The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.

The membrane skeleton of erythrocytes. A percolation model

The spectrin network on the cytoplasmic surface of the erythrocyte membrane is modeled as a triangular lattice of spectrin tetramers. This network obstructs lateral diffusion of proteins and provides mechanical reinforcement to the membrane. These effects are treated in a systematic and unified manner in terms of a percolation model. The diffusion coefficient is obtained as a function of the fraction of normal spectrin tetramers for both static and fluctuating barriers. The elasticity of the network is calculated as a function of the fraction of normal spectrin and the ratio of bending to stretching energies. For static barriers, elasticity and lateral diffusion are incompatible: if a network is connected enough to be elastic, it is connected enough to block long-range lateral diffusion. The elasticity and the force required for mechanical breakdown go to zero at the percolation threshold; experimental evidence suggests the existence of a stability threshold at or near the percolation threshold. The model is qualitatively applicable to other cells with membrane skeletons, such as epithelial cells, in which localization of membrane proteins is essential to differentiation.

Differing erythrocyte membrane skeletal protein defects in alpha and beta thalassemia.

Thalassemic red cells show irregular morphology and maldistribution of glycoproteins and sialic acids. These changes are compatible with damage to the red cell membrane skeleton. To test this possibility, we systematically studied the interconnections of skeletal proteins in patients with a form of alpha thalassemia (HbH disease), in patients with beta thalassemia intermedia, and in normal individuals. Alpha- and beta-thalassemic spectrin functions normally in spectrin self-association, binding to normal inside-out vesicles (IOVs), and binding to actin in the presence and absence of normal protein 4.1. Binding of normal spectrin to beta: thalassemic IOVs is normal but alpha-thalassemic IOVs are defective and bind only half the normal amount of spectrin (66 +/- 5 vs. 120 +/- 16 micrograms spectrin dimer/mg IOV protein, respectively). A different defect is detected in beta thalassemia, in which protein 4.1 shows markedly reduced ability (48 +/- 7% of normal) to enhance the binding of normal spectrin to actin and a decreased ability to bind normal spectrin in a binary interaction, compared with normal protein 4.1 (24 +/- 1 and 43 +/- 1 micrograms protein 4.1/mg spectrin, respectively). As no quantitative deficiency of beta-thalassemic protein 4.1 is detected, we assume an acquired lesion is present, which affects about half of the protein 4.1 molecules. These findings indicate that specific, localized, yet different defects exist in the skeletal proteins of alpha- and beta-thalassemic red cells. The different molecular lesions imply that the mechanism of hemolysis and probably the interaction of unpaired globin chains with the membrane differs in the two diseases.

Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis.

Whereas marked variations in the clinical manifestations of hereditary spherocytosis have long been recognized, we have only recently begun to define the molecular basis for this heterogeneity. An important unanswered question is whether decreased spectrin results in reduced membrane mechanical stability, and if this reduction in membrane mechanical stability can be related to in vivo surface area loss. Using the ektacytometer, we quantitated membrane surface area and stability in erythrocytes from 18 individuals with hereditary spherocytosis and deficiencies of spectrin (30-80% of normal spectrin level). Membrane mechanical stability was reduced and the magnitude of the reductions correlated with the spectrin content. Moreover, the reductions in mechanical stability correlated with in vivo loss of membrane surface area. These data indicate that decreased spectrin content results in reduced membrane mechanical stability and surface area loss in vivo. We conclude that partial deficiencies of spectrin, reductions in membrane mechanical stability, and loss of membrane surface area are directly related and are major features determining the heterogeneous clinical manifestations of hereditary spherocytosis.

Interactions of spectrin in hereditary elliptocytes containing truncated spectrin beta-chains.

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.

Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.

Molecular Determinants of Clinical Expression of Hereditary Elliptocytosis and Pyropoikilocytosis

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.