Spectrin self-association site: characterization and study of beta-spectrin mutations associated with hereditary elliptocytosis.

Most of hereditary elliptocytosis (HE) cases are related to a spectrin dimer (SpD) self-association defect. The severity of haemolysis is correlated with the extent of the SpD self-association defect, which itself depends on the location of the mutation regarding the tetramerization site. This site is presumed to involve the first C helix of the alpha chain and the last two helices, A and B, of the beta chain to reconstitute a triple helical structure (A, B and C), as observed along spectrin. Using recombinant peptides, we demonstrated that the first C helix of the alpha chain and the last two helices of the beta chain alone are not sufficient to establish interactions, which only occurred when a complete triple-helical repeat was added to each partner. One adjacent repeat is necessary to stabilize the conformation of both N- and C-terminal structures directly involved in the interaction site and is sufficient to generate a binding affinity similar to that observed in the native molecule. Producing peptides carrying a betaHE mutation, we reproduced the tetramerization defect as observed in patients. Therefore, the betaW2024R and betaW2061R mutations, which replace the invariant tryptophan and a residue located in the hydrophobic core, respectively, affect alpha-beta interactions considerably. In contrast, the betaA2013V mutation, which modifies a residue located outside any presumed interacting regions, has a minor effect on the interaction.

Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin.

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two-thirds of HE individuals we identified molecular defects, primarily those in erythrocyte alpha-spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte beta-spectrin (beta-W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the alpha III domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the alpha-spectrin gene, including the alpha III domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.

Inhibition of phospholipase D activity by fodrin. An active role for the cytoskeleton.

Phospholipase D (PLD) is a major enzyme implicated in important cellular processes such as secretion and proliferation. The knowledge of its regulation is essential to understand the control of these phenomena. Several proteins activating PLD have been described in the last years. In this report, we chromatographed bovine brain cytosolic proteins to identify fodrin, the non-erythroid spectrin, as the first described inhibitor of PLD. A cytosolic fraction with an inhibitory effect on PLD activity loses its capacity after immunoprecipitation of fodrin. Moreover, at 1 nM, purified fodrin blocks fully and quickly PLD activity, whatever the stimuli used. In contrast, fodrin has no effect on adenylate cyclase activity. Fodrin-analogous proteins like dimeric or tetrameric erythroid spectrin have the same inhibitory effect on PLD, at higher concentrations. Other cytoskeletal proteins, actin and vimentin, are inefficient on PLD inhibition. The mechanisms implicated in PLD modulation such as post-translational modifications of fodrin and the role of small G-proteins on the cytoskeleton regulation are discussed. In conclusion, this study reveals that fodrin is involved in the control of PLD activity, suggesting that the cytoskeleton could have an active role in control of secretion and proliferation.

Molecular basis of clinical and morphological heterogeneity in hereditary elliptocytosis (HE) with spectrin alpha I variants.

The impaired ability of spectrin dimers to self-associate into tetramers is one of the most frequent defects associated with hereditary elliptocytosis (HE) and its more serious form, hereditary pyropoikylocytosis (HPP). We previously described four proteic variants of the spectrin (Sp) alpha I tryptic domain associated with the Sp dimer self-association defect (Sp alpha I/78, Sp alpha I/74, Sp alpha I/65, Sp alpha I/46 variants). Following the characterization of proteic variants, genomic molecular defects were identified and most of the mutations appeared to lie either in or near the self-association site, i.e. in the alpha I tryptic domain or in the beta I tryptic domain. The clinical severity of these different mutations varies considerably and ranges from asymptomatic to severe haemolytic disease such as in heterozygous HPP patients and in some homozygous HE patients. Studies of 113 patients from 61 HE families showed a correlation among parameters and showed which factors modulate the clinical expression of the molecular defect. Our analysis indicated that the clinical expression was directly correlated with the severity of the spectrin dimer self-association defect as evaluated by the increase in the Sp dimer percentage found in the 4 degrees C extract. A critical threshold of 40-50% of unassembled Sp dimer was determined; above that, patients exhibited severe haemolysis requiring splenectomy. The percentage of Sp dimer depends, in turn, on two factors: (i) the nature of the variant in relation to the position of the mutation versus the tetramerization site; (ii) the relative amount of mutant spectrin present in the membrane (ranging from 15% to 80% in heterozygous patients). As for the severity of haemolysis, the ghost mechanical stability to shear stress, as measured by ektacyometer, was also found to depend on the Sp dimer self-association defect. In contrast, the decrease in erythrocyte deformability was not related to the amount of unassembled Sp dimer but appeared to be correlated with the amount of mutant spectrin whatever the variant. Concerning erythrocyte morphology and the number of elliptocytes, the Sp alpha I/65 variant appears to be the most 'elliptocytogenic' variant, indicating that erythrocyte shape abnormality is not linked to the Sp dimer self-association defect.

Heterogeneous phosphorylation of erythrocyte spectrin beta chain in intact cells.

Human erythrocyte spectrin is an alpha beta heterodimer which forms tetramers by self-association. This association involves the N-terminal region of the alpha chain and the C-terminal region of the beta chain. The latter contains a cluster of four phosphorylation sites (one phosphothreonine and three phosphoserine residues). The role of this phosphorylation is as yet unknown. We show in this paper that the spectrin beta chain occurs in the cell in subpopulations differing in the degree of occupancy of their phosphorylation sites: 32P peptide maps obtained by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage revealed the presence of six components with apparent molecular masses of 17.5 kDa, differing in their isoelectric points; this is most simply interpreted as reflecting the presence of six exchangeable phosphorylation sites in the spectrin beta chain, rather than four as had been supposed. When the alpha beta dimers were partly dissociated by urea, the most highly phosphorylated fraction of the beta chain was found in the undissociated dimers. This high specific activity in the undissociated dimer reflected multiple phosphorylated sites, as revealed by NTCB cleavage. The dephosphorylation or the hyperphosphorylation of spectrin beta chains did not modify the equilibrium between dissociated and undissociated spectrin dimers in the presence of urea. However, the data revealed the existence of two spectrin dimer populations in respect to phosphate turnover and spectrin dimer dissociation.

Spectrin beta Tandil, a novel shortened beta-chain variant associated with hereditary elliptocytosis is due to a deletional frameshift mutation in the beta-spectrin gene.

An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3' ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

Elliptocytosis-associated spectrin Rouen (beta 220/218) has a truncated but still phosphorylatable beta chain.

Spectrin Rouen (beta 220/218) is a novel variant, carrying a shortened beta chain with an apparent molecular weight of 218 kDa. It was detected in a French family. All affected members suffered from haemolytic hereditary elliptocytosis. As other shortened beta chain variants described before, the beta Rouen chain is truncated at its carboxyl terminus. Spectrin Rouen is associated with a defect in spectrin dimer self-association and with an abnormally high amount of the alpha I 74 kDa peptide following partial tryptic digestion. Dimer reconstitution experiments from normal and abnormal purified Sp subunits indicated that the increased alpha I 74 kDa fragment is induced by the altered beta chain. However, spectrin Rouen is different from other mutants with a truncated beta chain in several respects: its amount is low (less than 10%) and the spectrin dimer self-associated defect is mild. Critically, the beta Rouen chain has retained the ability of undergoing phosphorylation, even though it is modified in its C-terminal region. These results, compared to those obtained with beta 220/214 spectrin Le Puy and beta 220/216 spectrin Nice, allowed better localization of the beta chain sites that can be phosphorylated by a membrane-bound casein kinase.

Abnormal tryptic peptide from the spectrin alpha-chain resulting from alpha- or beta-chain mutations: two genetically distinct forms of the Sp alpha I/74 variant.

Limited tryptic digestion of native spectrin (Sp) has revealed several variants in hereditary pyropoikilocytosis (HPP) and in a subset of patients with hereditary elliptocytosis (HE). In most cases, tryptic peptide corresponding to the alpha I (N-terminal) 80 kD domain is wholly or partially replaced by smaller fragments. These variants are provisionally designated according to the molecular weight of the most prominent new peptide. Partial amino acid sequences of the abnormal peptides and DNA analysis of the alpha-spectrin gene have shown that most variants result from substitution or insertion of an amino acid in the alpha I-domain. However, similar investigations did not detect any such abnormality in the spectrin alpha I-domain of an HE black kindred with one of the spectrin variants called Sp alpha I/74. In this kindred, restriction fragment length polymorphism studies and transmission of the genetic polymorphism relative to the alpha II-domain excluded the involvement of the alpha-chain in the pathological process. To ascertain whether the abnormal alpha I 74 kD peptide might be caused by a beta-chain mutation, we reconstituted hybrid dimers combining normal and HE Sp-chains. The tryptic peptide patterns of spectrin hybrid dimers containing HE alpha-chain and control beta-chain showed a normal 80 kD tryptic product. In contrast, the hybrid dimer containing normal alpha-chain and HE beta-chain gave rise to increased 74 kD peptide at the expense of the 80 kD, demonstrating that the mutation in this family resides in the beta-chain. The same method was used to show that in two other unrelated white kindreds, the elevated 74 kD peptide arose from a Sp alpha-chain defect. Thus an alteration in tryptic susceptibility within the N-terminal domain of the spectrin alpha-chain can be directed by a mutation in the beta-chain. The hybridization technique affords a definitive means of distinguishing between alpha- and beta-chain mutants.

Hereditary pyropoikilocytosis and elliptocytosis in a white French family with the spectrin alpha I/74 variant related to a CGT to CAT codon change (Arg to His) at position 22 of the spectrin alpha I domain.

We describe a white French family in which 12 subjects presented with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP). Eight of these subjects were shown to be heterozygous for a spectrin (Sp) alpha I/74 variant, as demonstrated by analysis of partial tryptic digestion fragments of spectrin. This abnormal peptide pattern was associated with a decreased ability of Sp dimers to self-associate. In this kindred, in which four generations were available for study, the clinical expression varied from mild HE to HPP with an intermediate status of hemolytic HE. The severity of the disease appeared to be correlated both with the estimated amount of variant Sp (42% to 65%) and the excess of Sp dimers found in the membrane (30% to 51%, with a normal value of 3.7% +/- 1.6%). Reassociation studies using isolated Sp alpha and beta chains from an affected patient and an unaffected control subject showed that the Sp alpha I/74 Kd abnormal tryptic peptide resulted from a defect in the Sp alpha chain. Partial amino acid sequencing showed that the Sp alpha I/74 Kd peptide resulted from cleavage at lysine residue 42 of the Sp alpha I/80 Kd domain. Knowledge of the exon/intron organization of the human alpha Sp gene allowed us to amplify by the polymerase chain reaction the second exon of the alpha Sp gene in total cellular DNA of the HPP proposita. The amplified fragment was subcloned and sequenced. We found a G to A base substitution in the 22nd codon (CAT for CGT), which changes the normal arginine to a histidine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in six other HE and HPP members of the family. The identification of this mutation at the DNA level confirmed the transmission of the same molecular defect in Sp through four generations but with different patterns of clinical expression.

Severe recessive poikilocytic anaemia with a new spectrin alpha chain variant.

A severe neonatal haemolytic anaemia was observed in a propositus from the West Indies. Frequent blood transfusions were required until complete splenectomy was carried out. Blood smears showed predominant poikilocytosis with spherocytes and microcytes as observed in hereditary pyropoikilocytosis. Erythrocytes were completely fragmented after incubation at 45 degrees C. The two asymptomatic parents had normal haematological profiles. The erythrocyte membrane electrophoretic patterns of the splenectomized propositus and her parents were normal. The propositus had a moderate defect in the spectrin (Sp) dimer self-association. Limited tryptic digestion of the propositus' Sp under standard conditions (0 degrees C, 20 h, enzyme-substrate ratio of 1/100) revealed an increased sensitivity to tryptic digestion. The major features detected by one- and two-dimensional electrophoresis gels of Sp tryptic digests were the absence of high molecular weight peptides from the Sp alpha II (48 kDa and 35 kDa peptides) and Sp alpha III (52 kDa peptide) domains with increased amounts of the lower molecular weight peptides from the Sp alpha II and Sp alpha III (29 kDa peptide and 47 kDa peptide) domains respectively. Kinetic studies of Sp tryptic digestion (10 min to 36 h) confirmed the increased tryptic susceptibility of Sp. Immunodetection with specific anti-alpha-chain domain antibodies showed that the highest molecular weight peptides from the alpha II and alpha III domains are released early in digestion, but disappear quickly, with an increase in their corresponding smaller peptides. The molecular weights of the peptides corresponding to the 48 kDa and 35 kDa peptides from the alpha II domain are slightly modified. Peptides from the alpha I and alpha IV domains showed no significant abnormalities. The Sps of both parents were normal. These results indicate that the patient has a novel Sp alpha chain defect, which is probably located in the region of the alpha II domain which adjoins the alpha III domain.

Sp alpha I/78: a mutation of the alpha I spectrin domain in a white kindred with HE and HPP phenotypes.

Limited tryptic digestion of spectrin (Sp) from seven related individuals manifesting hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) phenotypes revealed the presence of a novel peptide with a molecular weight of 78 Kd and a concomitant decrease in the alpha I domain (80-Kd peptide), which is the domain involved in the dimer self-association process. Sp from the normal members of this white family exhibited a normal peptide pattern, as compared with controls. The abnormal peptide pattern was associated with a decreased ability of Sp dimer to self-associate. In this kindred in which three generations were available for study, the clinical manifestations were quite variable and ranged from the asymptomatic HE carrier state to hemolytic HE or to severe anemia requiring splenectomy. The severity of the disease appeared to be correlated both with the amount of mutant spectrin (31% to 69%) and with the excess of the Sp dimer found in the membrane (26% to 60%, compared with a normal value of 5.6% +/- 2.2%). Partial amino acid sequencing showed that the alpha I/78-Kd peptide resulted from cleavage at lysine residue 10 of the alpha I/80-Kd domain. Knowledge of the exon/intron structure of cloned genomic DNA encoding the alpha I domain allowed us to amplify in vitro a DNA fragment containing the third exon of the alpha-spectrin gene. The amplified fragment was subcloned and sequenced. A G to T transversion was found in the 39th codon (AGT for AGG), which changed the normal arginine to a serine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in three other HE members of the family (the propositus mother, brother, and sister).

[Hereditary elliptocytosis in West Africa: frequency and repartition of spectrin variants]. / L'elliptocytose héréditaire en Afrique de l'Ouest: fréquence et répartition des variants de la spectrine.

Hereditary Elliptocytosis (HE) is a hemolytic disorder inherited as autosomal-dominant trait and characterized by elliptically shaped erythrocytes. Preliminary studies in France have showed a high proportion of HE patients of black extraction (West Africa and Antilles). In order to confirm this prevalence, we made a systematic search for HE in West Africa: Benin, Burkina Faso, Ivory Coast, Togo. The diagnosis of elliptocytosis was established by the observation of a high percentage (greater than 70%) of characteristic regular and symmetric elliptic red cells after fixation in 0.3% glutaraldehyde saline buffer. The diagnosis of HE was confirmed by cytological studies of related members and/or the discovery of a well defined molecular variant of spectrin, the main protein of erythrocyte membrane skeleton. We found: in Abidjan centre 6 HE out of 1,000 subjects representative of main ethnic groups; in Lome Centre 6 cases out of 750 subjects originated from the South or Central areas of Togo; in Cotonou Centre 5 cases out of 1,000 subjects originated from the South area of Benin; in Bobo Dioulasso centre 6 HE out of 700 subjects. From this multicentre studies HE appears roughly 10 times more frequent in West Africa than in Europe or USA where incidence was estimated at between 2.5 and 5 cases per 10,000. Tryptic digestion of spectrin revealed that: 10 patients from different ethnic groups have the most frequent variant found in our laboratory (21 kindreds) and named spectrin alpha I/65. Five cases originated from limited areas in the South of Benin and Togo and related to closed ethnic groups have the variant Sp alpha I/46.

Hereditary pyropoikilocytosis and elliptocytosis in a Caucasian family. Transmission of the same molecular defect in spectrin through three generations with different clinical expression.

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia characterized by a material instability of the red cell membrane leading to cell fragmentation. This fragility may be correlated with functional and structural defects of spectrin. Most HPP patients have been black. We now report three HPP patients from a Caucasian family, the proposita and her two maternal uncles. The proposita's mother and daughter presented mild type I hereditary elliptocytosis (HE), while the proposita's father was clinically and hematologically normal. Our studies revealed a defective ability of spectrin to self-associate, resulting in an excess of spectrin dimer in 4 degrees C extracts in the three HPP patients and to a similar extent in HE relatives. Limited tryptic digestion of spectrin showed a molecular variant in the alpha I domain as expressed by a decreased amount of 80,000-dalton peptide with a concomitant increase in the 74,000-dalton peptide. Investigations in the proposita's father revealed no abnormalities of the erythrocyte membrane. The co-transmission of HPP and HE phenotypes in the same lineage might suggest variability in the clinical expression of the same molecular defect and lead us to discuss the hypothesis of a double heterozygosity in HPP patients.

Prenatal diagnosis of hereditary elliptocytosis with molecular defect of spectrin.

Hereditary elliptocytosis (HE) is, in the heterozygous state, a common mild congenital hemolytic disease. In contrast, homozygous elliptocytosis is a severe transfusion-dependent hemolytic anemia. The major determinant of red cell membrane shape and stability is a two-dimensional proteinaceous meshwork named membrane skeleton. Spectrin, the most important protein of the membrane skeleton, is basically a heterodimer composed of alpha and beta chains. Within the membrane, spectrin dimers self-associate to form tetramers. In type I HE spectrin dimer self-association is defective and an excess of spectrin dimer is present in the patient's red cell membranes. The defective self-association is often correlated with an abnormality of the spectrin alpha chain which is depicted by limited tryptic digest of spectrin. In a family previously studied by us (Dhermy et al., 1984), the search for a spectrin defect in the red cells of the fetus of the pregnant mother was indicated for the following reasons: the diagnosis of heterozygous type I HE with the same spectrin variant had been made in the mother as well as in the father. Moreover, homozygous HE had been recognized in one of the children born two years previously with a persistent and severe transfusion dependent hemolytic anemia. Preliminary studies of normal fetal erythrocytes at twenty weeks gestation have shown that fetal and adult spectrin molecules are identical. The results obtained in the fetus at risk allowed us to diagnose type I HE (though elliptocytes were not present in the blood) for the following reasons: (i) erythrocyte deformability was decreased (ii) spectrin self-association was defective with an excess of dimer species in the membrane (iii) limited tryptic digest of spectrin showed the same abnormal pattern as seen in the heterozygous mother, with a decrease in the 80,000-dalton peptide and a concomitant increase in the 74,000-dalton peptide. The heterozygous state, strongly suspected on the tryptic digest pattern of fetal spectrin, was confirmed when the mother gave birth to a baby who did not have hemolytic anemia during the first 18 months of life.

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis.

Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, alpha and beta. In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin alpha chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp alpha I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (alpha 2I/65-beta 2) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.

Double inheritance of an alpha I/65 spectrin variant in a child with homozygous elliptocytosis.

Hemolytic anemia with red cell fragmentation, poikilocytosis, and elliptocytosis was discovered in a 6-week-old black infant. Both parents and a brother of the propositus had compensated mild Hereditary Elliptocytosis (HE). Elliptocytosis was prominent in the proband's father with the presence of numerous rod-shaped cells whereas, in the proband's mother, elliptocytosis was less marked and cells were less elongated than in the father. The proband's red cells fragmented at 45 degrees C instead of 49 degrees C for control cells. Both the parents' and brother's red cells fragmented at 47 degrees C. The deformability of the proband's red cells was markedly reduced when measured with the ektacytometer; the red cells of both the proband's parent and brother exhibited an intermediate decrease in red cell deformability. Spectrin self-association was defective in the propositus as well as in his parents and brother. Limited tryptic digestion of the proband's spectrin, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed a complete absence of the normal 80,000 dalton alpha I domain and the presence of an abnormal 65,000 dalton peptide. Two-dimensional isoelectric focusing/SDS-PAGE of limited tryptic digests of spectrin from both the proband's parents and brother revealed a decrease in the normal 80,000 alpha I domain and the presence of the 65,000 peptide variant. On the basis of biochemic studies performed on the patients' spectrin, we concluded that the proband had homozygous HE, having inherited the structural defect of spectrin present in a heterozygous state in each of his parents. On a clinical and morphologic level, homozygous HE imitates two other forms of congenital hemolytic anemia associated with a spectrin self-association defect: HE with pycnocytosis in infancy and Hereditary Pyropoikilocytosis. This report emphasizes the importance of confronting clinical and rheological as well as biochemical investigations in studying and discussing different entities.

Hereditary elliptocytosis: clinical, morphological and biochemical studies of 38 cases.

We report clinical, morphological and biochemical studies performed on 38 cases of hereditary elliptocytosis (HE). The major determinant of membrane shape and stability is a proteinaceous meshwork named membrane skeleton, composed mainly of spectrin, actin, protein 4.1 and ankyrin. Spectrin is a heterodimer composed of two chains alpha and beta. Two spectrin dimers associate head to head to form a tetramer. Spectrin tetramers are cross-linked by actin and protein 4.1 to form the skeletal meshwork. We observed two types of membrane defects in the 38 patients studied: 24 patients (13 kindreds) exhibited spectrin self-association defect (type I HE) and 14 patients (6 kindreds) displayed deficiency in protein 4.1. A mutation in the spectrin chain was mostly found in the cases of type I HE. These mutations were depicted on tryptic digest patterns of spectrin. Three pathological variants were thus identified and characterized by the appearance of an abnormal peptide, with a molecular weight of either 74,000 or 65,000, or 46,000 daltons. In one family, the spectrin self-association defect was related to a shortened spectrin beta chain. Deficiency in protein 4.1 was found in 14 patients by means of polyacrylamide gel electrophoresis of red cell membranes. In 12 heterozygous cases of HE, the decrease in the amount of protein band 4.1 was between 40% and 50%. In 2 homozygous HE cases, protein band 4.1 was totally absent. Immunoelectrotransfer blots of red cell membrane proteins using a monoclonal antibody against protein 4.1 allowed characterization of additional bands in two families. In some cases variations in the amount of glycophorin C were noticed. Comparative studies of the two types of membrane abnormalities in HE clearly showed the absence of correlation between clinical, morphological phenotypes, and specific molecular etiology. However, all HE patients with protein 4.1 deficiency were caucasian and most of the type I HE were of black extraction. A study of red cell deformability using an ektacytometer revealed that the cell deformability under isotonic conditions was decreased in all HE patients. When the deformability was studied as a function of the osmolality of the suspending medium, the curve obtained had a trapezoid shape. This typical profile appeared to be constant in type I HE. We showed that the molecular abnormalities of the spectrin alpha chain, found in most type I HE correlated well with the functional spectrin defect.(ABSTRACT TRUNCATED AT 400 WORDS)

A new abnormal variant of spectrin in black patients with hereditary elliptocytosis.

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer-dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000-dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self-association in HE.

Pathologic and nonpathologic variants of the spectrin molecule in two black families with hereditary elliptocytosis.

Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE. Defective dimer-dimer association was present in all patients. Limited tryptic digestion of spectrin and subsequent analysis by one- and two-dimensional electrophoresis revealed a similar and reproducible decrease in the 80,000-dalton peptide (alpha I domain) and the concomitant appearance of a 46,000-dalton peptide. All the patients had the polymorphism of the spectrin alpha II domain commonly observed in black populations. In addition, modifications relative to the alpha III domain were detected; similar variants were found in one black control subject out of 136 and are likely related to a genetic polymorphism of the alpha III domain. No differences were observed between the peptide patterns in the infant with poikilocytosis and those of his HE sister and mother.