South-East Asian ovalocytosis among the population of the Highlands of Madagascar: a vestigé of the island's settlement.

In the Madagascar Highlands, 0.76% of children from 168 random primary schools, and 19 of 150 families from 3 villages, had oval-shaped erythrocytes. Most harboured the deletion in the band 3 gene characteristic of South-East Asian ovalocytosis. This genetic trait supports the Indonesian origin of the Madagascar settlement.

Differential subcellular localization of hZip1 in adherent and non-adherent cells.

Two human divalent cation transporters of the ZIP family, hZip1 and hZip2, homologous to Irt1 (Arabidopsis thaliana), the first identified member, have been described. They were shown by transfection into K562 cells to be localized at the plasma membrane and to mediate zinc uptake. Here we report a differential subcellular localization of hZip1 according to cell type. By transient expressions of EGFP-hZip1, FLAG-tagged or native hZip1, we observed that hZip1 has a vesicular localization in COS-7 cells or in several epithelial cell lines, corresponding partially to the endoplasmic reticulum. Using anti-hZip1 antibodies, we confirmed the intracellular localization of the endogenous protein in PC-3, a prostate cancer cell line.

Pleiotropic syndrome of dehydrated hereditary stomatocytosis, pseudohyperkalemia, and perinatal edema maps to 16q23-q24.

Dehydrated hereditary stomatocytosis (DHS) is a rare genetic disorder of red cell permeability to cations, leading to a well-compensated hemolytic anemia. DHS was shown previously to be associated in some families with a particular form of perinatal edema, which resolves in the weeks following birth and, in addition, with pseudohyperkalemia in one kindred. The latter condition was hitherto regarded as the separate entity, "familial pseudohyperkalemia." DHS and familial pseudohyperkalemia are thought to stem from the same gene, mapping to 16q23-q24. This study screened 8 French and 2 American families with DHS. DHS appeared to be part of a pleiotropic syndrome in some families: DHS + perinatal edema, DHS + pseudohyperkalemia, or DHS + perinatal edema + pseudohyperkalemia. If adequately attended to, the perinatal edema resolved spontaneously after birth. Logistic regression showed that increased mean corpuscular volume and mean corpuscular hemoglobin concentration were the parameters best related to DHS. In patients in whom cation fluxes were investigated, the temperature dependence of the monovalent cation leak exhibited comparable curves. Specific recombination events consistently suggested that the responsible gene lies between markers D16S402 and D16S3037 (16q23-q24). The 95% confidence limits (Z(max) >/= 3.02) spanned almost the complete 9-cM interval between these 2 markers.

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins.

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, alpha-and beta-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS-CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS-CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.

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.

Portal vein thrombosis after splenectomy for hereditary stomatocytosis in childhood.

UNLABELLED: Portal vein thrombosis is a rare but potentially lethal complication in children requiring splenectomy. We report on a 15-year-old boy with a dehydrated hereditary stomatocytosis, who underwent splenectomy and presented a postoperative partial portal vein thrombosis. With prompt heparin therapy, neither propagation of the thrombus nor further cavernous transformation in the following occurred 6 years. CONCLUSION: Recent data suggest that hereditary stomatocytosis carries a high risk of thrombotic complications, especially after splenectomy. This procedure, the benefit of which is limited in this condition, should therefore be strongly avoided.

Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin.

We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the alpha-chain and the C-terminus of the beta-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the beta-chain and one helix from the N-terminus of the alpha-chain. Three mutant beta-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The alpha- and beta-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded beta-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phenomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wildtype alpha- and beta-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in alpha-helicity. From continuous-variation profiles an association constant in the range 1-2 x 10(6) M-1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin beta-chain. Of the three mutations in the beta-chain fragment, one (an Ala-->Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the alpha-chain fragment, whereas Trp-->Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations.

Polymorphisms of the tissue factor pathway inhibitor (TFPI) gene in patients with acute coronary syndromes and in healthy subjects : impact of the V264M substitution on plasma levels of TFPI.

-Mutations of the gene encoding tissue factor pathway inhibitor (TFPI), an inhibitor of TF-induced activation of the coagulation cascade, were screened for in 130 patients and 142 healthy controls to determine whether these variants contribute to acute coronary syndromes or modify plasma TFPI levels. The following 3 new polymorphisms were identified: 384T-->C in exon IV, which does not change the corresponding amino acid (tyrosine 57); -33C-->T in intron 7 (the T/T, C/T, and C/C genotypes were found in approximately 50%, 40%, and 10% of subjects in both groups); and 874G-->A in exon IX (GTG-->ATG), which predicts a valine to methionine change (V264M) in the carboxy-terminus tail of TFPI. The V264M polymorphism was found in 9.2% of the cases and 4.9% of the controls; the associated odds ratio (OR) for acute coronary syndromes was 2.0 (95% confidence interval [CI], 0.7 to 5.1). The OR increased to 3.6 (95% CI, 0.8 to 15.7) and 3.2 (95% CI, 0.9 to 11.8) in nonsmokers and patients without other risk factors, respectively. The possible link between the V264M polymorphism and coronary heart disease was checked in a large case-control study of myocardial infarction (Etude Cas-Témoins de l'Infarctus du Myocarde [the ECTIM Study]). The results showed no link between the V264M polymorphism and coronary syndromes. Interestingly, however, 5 patients heterozygous for the V264M polymorphism had significantly lower plasma TFPI levels than did 13 patients with the most common genotype. Although our present results do not support an association between TFPI polymorphisms and acute coronary syndromes, the possibility that 1 of them, especially the exon IX polymorphism, is associated with subtypes of myocardial infarction or to evolutive particularities that were not assessed in this study, cannot be excluded and is currently being evaluated.

The red blood cell band 3 variant (band 3Biceêtrel:R490C) associated with dominant hereditary spherocytosis causes defective membrane targeting of the molecule and a dominant negative effect.

Hereditary spherocytosis (HS), a common human inherited haemolytic anaemia, is associated with partial deficiency of different erythrocyte membrane proteins. In a subset of dominant HS, a partial membrane expression deficiency of band 3, the erythrocyte anion exchanger (AE1), have previously been characterized, and several mutations in the band 3 gene have been found: amino acid substitutions at conserved positions in the membrane domain, nonsense and frameshift mutations. In HS patients bearing missense mutations, the mutated transcript was present, whereas only the normal transcript was found in HS patients with frameshift mutations. In the former group, the membrane expression deficiency of band 3 was significantly more important than that observed in the latter group of HS patients with frameshift mutations, suggesting that missense mutations may have a dominant negative effect. In the present study, transient and stable transfections of K562 and COS-7 cells were used to demonstrate, by immunoblots of cell lysates and immunofluorescence studies, that the band 3 membrane domain bearing the R490C mutation (band 3Bicetrel) is not targeted to the plasma membrane and is retained in the endoplasmic reticulum. Transient cotransfections of K562 cells with plasmid coding for the normal membrane domain of band 3, together with increasing amounts of plasmid coding for the mutated R490C membrane domain, demonstrated that the band 3 mutant polypeptide exerts a dominant negative effect on the plasma membrane targeting of the normal band 3.

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.

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.

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.

Red blood cell abnormalities in hereditary elliptocytosis and their relevance to variable clinical expression.

Marked variations are seen in the clinical manifestations of hereditary elliptocytosis (HE). To define the cellular alteration(s) that best reflect the variable expression of the disease, we evaluated the pathobiologic features of red blood cells in a series of 18 patients with HE, 15 persons from six families with HE as a result of defects in spectrin, and 3 persons from one family with HE caused by partial or total deficiency of protein 4.1. We found that decreased cellular deformability is a distinguishing feature of red blood cells in all patients studied. Comparison of volume and hemoglobin content histograms of red blood cells and reticulocytes revealed that cell fragmentation is a feature of mature red blood cells. The extent of red blood cell fragmentation as reflected by increased percentage of microcytic red blood cells was the best indicator of the severity of hemolytic anemia. Furthermore, we found that the observed variations in cellular properties of HE red blood cells in different persons is the consequence of varying amounts of mutant protein assembled into the membrane. These findings enabled us to define the mechanistic basis for cellular changes in this red blood cell membrane disorder better and also to obtain insight into the cellular basis for variable clinical expression.

Isochromosome 14q in refractory anemia.

Trisomy 14 in hematologic disease is a rare finding and is almost exclusively associated with myeloid cell lineage. We present a case of refractory anemia (MDS-RA) with the uncommon features of marked elliptocytosis and schistocytosis in the peripheral blood and isochromosome 14q. The analysis of the clinical outcome of this case and of others of myelodysplastic (MDS)/myeloproliferative syndromes with trisomy 14 as the sole abnormality suggests that it does not confer an unfavorable prognosis.

Heterogenous band 3 deficiency in hereditary spherocytosis related to different band 3 gene defects.

Among 80 hereditary spherocytosis (HS) kindreds studied using denaturing electrophoretic separation of solubilized eythrocyte membrane proteins, we recognized three prominent subsets: HS with isolated spectrin deficiency, HS with combined spectrin and ankyrin deficiency, and HS with band 3 deficiency These three subsets represent more than 80% of the HS kindreds studied. In this study, eight dominant HS kindreds with band 3 deficiency were investigated for band 3 mutations. In three of these kindreds, linkage analyses confirmed the band 3 gene as the culprit gene. In an attempt to identify the responsible mutations, denaturing gradient gel electrophoresis (DGGE) was used to explore the coding exons (exons 2-20) of band 3 gene. Five different mutations were found in the eight kindreds. In five kindreds we identified substitutions of highly conserved residues, positioned at boundaries of putative transmembrane segments: a C --> T substitution at codon 490 changed arginine (CGC) to cysteine (TGC) in three kindreds, a C --> T substitution at codon 837 changed threonine (ACG) to methionine (ATG) in two kindreds. In the sixth kindred a G deletion was found in a stretch of five G starting at position 1475, leading to a stop codon either at position 1527 or 1565. In the seventh kindred a T deletion at position 1600 resulted in a stop codon at position 1733 and in the last kindred a T deletion was identified at position 355, leading to a stop codon at position 447. The mutant transcript was present in HS patients bearing missense mutations, whereas only the normal transcript was found in HS patients with frameshift mutations. In the latter group the mean decrease in membrane band 3 content was significantly lower, leading to speculation that missense mutations may have some sort of dominant negative effect.

Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia.

An alpha-spectrin variant with increased susceptibility to tryptic digestion, alpha(II/47), was previously observed in a child with severe, recessively inherited, poikilocytic anemia. The molecular basis of this variant, spectrin St Claude, has now been identified as a splicing mutation of the alpha-spectrin gene due to a T --> G mutation in the 3' acceptor splice site of exon 20. This polypyrimidine tract mutation creates a new acceptor splice site, AT --> AG, and leads to the production of two novel mRNAs. One mRNA contains a 12 intronic nucleotide insertion upstream of exon 20. This insertion introduces a termination codon into the reading frame and is predicted to encode a truncated protein (108 kD) that lacks the nucleation site and thus cannot be assembled in the membrane. In the other mRNA, there is in-frame skipping of exon 20, predicting a truncated (277 kD) alpha-spectrin chain. The homozygous propositus has only truncated 277 kD alpha-spectrin chains in his erythrocyte membranes. His heterozygous parents are clinically and biochemically normal. This allele was identified in 3% of asymptomatic individuals from Benin, Africa.

Human erythroid spectrin alpha subunit and its SH3 domain are sensitive to acidic Plasmodium falciparum proteolytic activity.

Many proteases play a crucial role in the Plasmodium intraerythrocytic life cycle. Spectrin depletion, one of the major events involved in parasite release from the red blood cell, results from proteolytic activities associated with the presence of the intracellular parasite. Here, we describe a new acidic proteolytic activity from Plasmodium falciparum, whose target is the alpha-subunit of human spectrin. Immunoblotting experiments with antibodies specific for the tryptic peptides of the alpha-chain and in vitro proteolysis tests on recombinant peptides from different regions of the spectrin alpha subunit demonstrated that cleavage sites for the parasite proteolytic activity were localized within the SH3 motif of the alpha-chain sequence. Remarkably, this Plasmodium protease activity on spectrin SH3 substrate was unable to cleave the SH3 from fodrin, a non-erythroid spectrin.

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.