Spectrin beta-chain variant associated with hereditary elliptocytosis.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

[Anti-muscle actin and anti-erythrocyte spectrin in sera containing smooth muscle auto-antibodies (author's transl)]. / Mise en évidence d'anticorps anti-actine musculaire et anti-spectrine érythrocytaire dans des sérums possédant une activité anti-muscle lisse.

Using immunodiffusssion tests, the authors describe anti-muscle actin and anti-erythrocyte spectrin auto-antibodies in sera of patients with chronic active hepatitis. These results are compared with study of sera containing anti-striated muscle auto-antibodies and the meaning of these anti-spectrin antibodies is discussed.

Human erythrocytic calcium concentration in hemolytic anemia.

In order to elucidate the role of the Ca2+ accumulation in hemolytic process, erythrocytic calcium concentration has been studied by atomic absorption spectroscopy in 18 cases of congenital hemolytic anemias and in 19 cases of acquired auto-immune hemolysis. An increased erythrocytic Ca2+ level was observed in some, but not in all, congenital (10 cases) or acquired (6 cases) hemolytic anemias. It seems that the loss in erythrocytic deformability induced by calcium accumulation probably was not the common state leading to hemolysis.

Artefacts due to sodium dodecylsulfate polyacrylamide gel electrophoresis in the study of human erythrocyte membrane calcium binding protein.

Sodium dodecylsulfate polyacrylamide gel electrophoresis is not usable for identification of calcium binding protein from human erythrocyte membrane; Ca2+ binds to anionic sites of SDS and with 45Ca radioactivity bands appear in gels in absence of proteins.

[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.

Unclassified haemolytic anaemia with splenomegaly and erythrocyte cation abnormalities--a disease of the spleen?

An unclassified case of haemolytic anaemia with voluminous splenomegaly is reported. This anaemia was normocytic without any specific morphologic aspect of red blood cells (RBC); Coombs test was negative; the osmotic fragility was normal; the increased autohaemolysis was not affected by the presence of glucose; Hb studies were normal; no RBC enzyme deficiency was found; RBC lipids and membrane proteins were normal; there was a marked reduction in RBC survival with exclusive splenic uptake of erythrocytes. Before splenectomy, RBC cations and water content were abnormal: 1) the RBC water was decreased moderately; 2) the RBC sodium was about twice the normal mean with an increased 22Na turn-over; 3) the RBC potassium was markedly reduced and 42K influx was twice the normal mean; 4) the RBC calcium content was increased. Splenectomy was followed by rapid disappearance of haemolysis and RBC water and cation disturbances. Because of this extremely rapid disappearance after splenectomy the authors suggest this case of haemolytic anaemia could be a primary disease of the spleen.

Purification of erythrocyte protein 4.1 by selective interaction with inositol hexaphosphate.

Protein 4.1 is a multifunctional structural protein occupying a strategic position in the erythrocyte membrane. It is present in the erythrocyte membrane skeleton and in many nonerythroid cells. This report describes a novel method for purifying this protein based on its selective interaction with inositol hexaphosphate dimagnesium tetrapotassium salt. This interaction was discovered in the course of chromatography of high-salt extract of inside-out membrane vesicles on Procion orange MX-2R-Sepharose. The new procedure is simple and selective and produces protein 4.1 with better yield than that obtained with a previously published procedure. The purified protein 4.1 has the same immunoreactivity and the same alpha-chymotryptic digest profile as protein 4.1 purified by published methods and is fully functional in enhancing the interaction between F-actin and spectrin dimers.

Anti-spectrin in sera containing smooth muscle autoantibodies from patients with chronic active hepatitis.

Sera from patients with chronic active hepatitis containing anti-smooth muscle autoantibodies (SMA) react against rabbit muscle actin as well as human red cell spectrin. These anti-spectrin antibodies recognize the same antigen as rabbit anti-human spectrin antibodies and are not involved in the staining pattern given by SMA-containing sera tested with smooth muscle sections. These anti-spectrin antibodies probably recognize an antigenic structure common to both spectrin and another as yet undetermined molecule (which however is not likely to be myosin).

Hereditary spherocytosis with spectrin deficiency related to null mutations of the beta-spectrin gene.

Spectrin deficiency is the most common deficiency found in HS. It is heterogeneous in terms of clinical expression, inheritance (dominant or recessive) and underlying genetic defects (related to alpha- or beta-spectrin gene defects or secondary to ankyrin gene defects). We studied a sampling of French dominant HS families, selected after linkage analyses, and found the presence of mutations resulting in the silencing of the mutant beta-spectrin allele. In three HS families, one haploid set of beta-spectrin mRNA was undectectable. In two families, a deletion of 8 bases (leading to a frameshift and a premature stop codon) and a nonsense mutation were identified, respectively. In the third HS family, we were unable to characterize a relevant mutation but the loss of heterozygosity at the cDNA level suggested the presence of a null mutation of the beta-spectrin allele. Sequencing of the beta-spectrin gene has also uncovered several new polymorphisms in the coding region of the beta-spectrin gene which will be very useful for detecting loss of heterozygosity at the cDNA level and designating the beta-spectrin gene as the culprit one.

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.

Hereditary elliptocytosis with a spectrin molecular defect in a white patient.

According to recent works, hereditary elliptocytosis (HE) appears to be related in some instances, to a defective self-association of spectrin (type I HE). We report a new case of type I HE observed in a white patient. Study of limited tryptic digestion of a spectrin dimer showed modification of a peptide involved in the dimer self-association process.

Coinheritance of two alpha-spectrin gene defects in a recessive spherocytosis family.

We studied a recessive hereditary spherocytosis (HS) family from Norway in which all four children had haemolytic spherocytosis while spectrin (Sp) deficiency was detected in the proband. Molecular analysis demonstrated that all affected children had inherited the low expression alpha-Sp allele LEPRA (Low Expressed PRAgue) from the father. Haplotyping with a polymorphic dinucleotide repeat for the alpha-Sp gene (alphaVNTR) located in the 3' untranslated region of mRNA showed that all recessive children had inherited the same maternal alpha-spectrin allele. The paternal Sp-alphaLEPRA allele was found in cis of the polymorphic alpha-Sp Bughill allele (alphaBH) characterized by the A970D point mutation in the Sp alpha-chain. This mutation was identified on two-dimensional electrophoresis of Sp tryptic digests as an acidic shift of the alphaII tryptic domains (spots alphaIIa). Analyses of the relative expression of the paternal alpha-Sp Bughill polymorphism in the proband showed that the product of the maternal alpha-Sp gene is almost completely absent from the mature erythrocyte membrane. Comparative analysis between alphaVNTR PCR-amplified from genomic DNA and from cDNA showed that the maternal low expression alpha-Sp allele is associated with a decreased amount of mRNA. Results from molecular and biochemical studies showed that all the affected children of this family are compound heterozygous for two different low expression alpha-Sp alleles: an uncharacterized defective alpha-Sp allele on the maternal side and an alphaLEPRA allele tagged by the alphaIIa polymorphism on the paternal side.

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.

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.

[Congenital hemolytic anemia with erythrocyte thermal instability and defect of spectrin tetrameric polymerization. Study of erythrocyte deformability in a new case of hereditary pyropoikilocytosis using diffraction viscosimetry]. / Anémie hémolytique congénitale par instabilité thermique érythrocytaire et défaut de tétramérisation de la spectrine. Etude de la déformabilité érythorcytaire d'un nouveau cas de pyropoïkilocytose héréditaire par visco-diffractométrie.

Hereditary pyropoikilocytosis (HPP) is a rare congenital hemolytic anaemia observed so far in patients of black extraction. In many cases, the severity of the anaemia has led to early splenectomy, which uniformly improved the hematological conditions. The disease is characterized by extreme anisocytosis and poikilocytosis with erythrocyte fragmentation. The pathognomonic feature is the abnormal thermal sensitivity of red cells which fragment in vitro at 45-46 degrees C (instead of 49 degrees C in normal subjects). In the case reported here, erythrocyte fragmentation (which appears at 43 degrees C) and increased osmotic fragility are studied using the Ektacytometer. Observation of different erythrocyte fractions isolated by differential centrifugation shows the extreme heterogeneity of the erythrocyte population. Studies of erythrocyte membrane proteins confirm that the disease is related to defective dimer-dimer association of spectrin which is the major membrane skeletal protein. This molecular spectrin abnormality in accounting for the membrane instability is recognized to a lesser degree in the asymptomatic mother. This may be of use in the diagnosis of HPP which is difficult to establish in a transfusion-dependent infant.

Search for the candidate genes in dominant hereditary spherocytosis using linkage analysis.

Hereditary spherocytosis (HS) is an inherited hemolytic anemia characterized by the presence of dense spherocytic red cells. In HS patients, red cell membrane protein gel electrophoresis has identified different subsets of abnormalities: isolated spectrin deficiency, combined spectrin and ankyrin deficiency, band 3 deficiency. To direct the search for the molecular defect in 9 families with dominant HS, we developed microsatellite markers specific for the membrane protein encoding genes possibly involved in HS (alpha- and beta-spectrin, ankyrin and band 3 genes) and genotyped each family. In 5 families with isolated spectrin deficiency, the beta-spectrin gene was designated as candidate. In one family with combined spectrin/ankyrin deficiency, only the ankyrin gene was not excluded, whereas in the 3 HS families with band 3 deficiency, only the band 3 gene was not excluded. This work allowed development of a reliable methodology to search for candidate genes in HS and showed the frequent involvement of the beta-spectrin gene in HS with isolated spectrin deficiency.

A 5' splice region G-->C mutation in exon 3 of the human beta-spectrin gene leads to decreased levels of beta-spectrin mRNA and is responsible for dominant hereditary spherocytosis (spectrin Guemene-Penfao).

We studied a family with autosomal dominant hereditary spherocytosis (HS) associated with a mild spectrin deficiency. Linkage analysis using two microsatellite markers (D14S63 and D14S271) very close to the beta-spectrin gene (SPTB) showed that HS co-segregated with alleles of these microsatellite markers and the linkage between the marker and HS was statistically significant. The presence of a beta-spectrin protein polymorphism (beta-spectrin Vay; A1880V) in trans of the HS allele was not itself deleterious, but allowed the detection of decreased membrane expression of the spherocytic beta-spectrin allele in two HS-affected subjects. Direct sequencing of the coding exons of the beta-spectrin gene in one affected subject showed the presence of a G-->C transversion at the terminal nucleotide of exon 3, which did not change the leucine codon 100 (CTG-->CTC). The presence of the mutation was confirmed by restriction enzyme digestion at the DNA level in all affected SH members of the family. The G-->C mutation severely reduced the utilization of the 5' splice site and resulted in aberrant mRNA splicing with intron 3 retention.

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.

A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis.

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

Molecular defect of spectrin in the family of a child with congenital hemolytic poikilocytic anemia.

We present the study of a black family in which the proband suffered from a severe neonatal hemolytic anemia with poikilocytosis. Both the parents, sister's, and brother's proband were clinically normal. The presence of poikilocytes in proband led to a search for a red cell membrane skeleton defect. Owing to recent improvements in the erythrocyte membrane knowledge, it is now possible to approach the diagnosis by means of biochemical evaluation of both parents, even if they are asymptomatic. So, the first time discovery of a spectrin self-association defect in both parents allowed us to suspect double inheritance of this abnormality in the proband. A complete morphological and biochemical evaluation of the family allowed us to propound the diagnosis of heterozygous type I hereditary elliptocytosis (HE) for both parents and the sister and the diagnosis of homozygous type I HE for the proband owing to the following reasons: slight ovalocytosis was present in both parents and the sister; cell deformability ektacytometric studies gave the same profiles of curve as those observed in patients with HE. Defective spectrin dimer self-association found in both parents was also observed in the sister and proband, associated with the same abnormal spectrin digest pattern, namely a decrease in the amount of a 80,000-dalton peptide and a corresponding increase in a 74,000-dalton peptide. However, clinical presentation of the proband was consistent either with hereditary pyropoikilocytosis or homozygous hereditary elliptocytosis; erythrocyte thermal sensitivity studies in the proband could not be conclusive because of the presence of transfused cells. Both these diagnoses are discussed in detail.(ABSTRACT TRUNCATED AT 250 WORDS)