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.

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.

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.

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.

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.

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 nonsense mutation in the erythrocyte band 3 gene associated with decreased mRNA accumulation in a kindred with dominant hereditary spherocytosis.

We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.

Ethnic distribution of allele alpha LELY, a low-expression allele of red-cell spectrin alpha-gene.

Allele alpha LELY is a low-expression allele of erythroid spectrin alpha-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele alpha LELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious alpha-alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele alpha LELY in various ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele alpha LELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele alpha LELY appears to be an ancient and stable allele.

A variant of spectrin low-expression allele alpha LELY carrying a hereditary elliptocytosis mutation in codon 28.

Allele alpha LELY is a low-expression allele of the erythroid spectrin alpha-gene. It carries mutations in exon 40 (alpha V/41 polymorphism) and intron 45, respectively, and is associated with partial skipping of exon 46. The latter phenomenon is thought to impair the recruitment of alpha-chains by beta-chains, and would eventually account for the low-expression character. When it occurs in trans to an alpha-allele responsible for hereditary elliptocytosis (alpha HE allele; alpha HE/alpha LELY diplotype), allele alpha LELY enhances the severity of elliptocytosis. Because allele alpha LELY is widespread, we anticipated that it would occasionally carry HE determinants. These variants of allele alpha LELY will be designated alpha HE-LELY allele. The HE component was the known alpha 28 Arg-->His mutation. This alpha HE-LELY allele was investigated within the alpha HE-LELY/alpha LELY diplotype, a diplotype not described before. Except for the neonatal period, the presentation was mild. In a consistent manner, the alpha LELY component in cis of the alpha HE mutation counteracted the like component in trans.

Identification of three novel spectrin alpha I/74 mutations in hereditary elliptocytosis: further support for a triple-stranded folding unit model of the spectrin heterodimer contact site.

Six individuals with hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) from three unrelated families were evaluated. Defects in the ability of spectrin (Sp) to undergo self-association were present, and associated with increased recovery of the Sp alpha I 74-kD fragment after limited tryptic digestion (Sp alpha I/74 variant). Because mutations associated with the Sp alpha I/74 variant described to date have been localized to the 5' coding region of the alpha-Sp gene (exon 2) or at the 3' coding end of the beta-Sp gene (exon 30), the polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) method was used to detect mutations in these two regions. In one family with HE, an abnormal pattern of migration of PCR-amplified fragments containing exon 2 was observed, and led to the detection of a new mutation (Ile24Ser) in helix 3 of repeating segment alpha 1. In the two other families, an abnormal pattern of migration of PCR-amplified fragments containing exon 30 was observed in affected individuals, and sequencing led to the identification of two new mutations (Ala2023Val and Trp2024Arg) in helix 1 of repeating segment beta 17. The elliptogenic potential of these mutations emphasizes the importance of the conformational integrity of each of the three helices involved in the formation of the Sp heterodimer contact site, and will help identify critical amino acids involved in this interaction.

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.

Spectrin alpha IIa variant in dominant and non-dominant spherocytosis.

Several polymorphic mutations are located on the spectrin alpha-chain; among these the variant termed alpha IIa is characterized by an acid shift in the isoelectric point of the tryptic digest peptides 46 kDa and 35 kDa. In this variant a single amino acid substitution (alanine to aspartic acid) occurred at position 972 of the spectrin alpha-chain due to a point mutation (GCT to GAT) in the DNA. This variant, which seemed very rare in normal people, could be related to the recessive form of hereditary spherocytosis (HS) and could be absent in the dominant form of the disease. We have studied the alpha IIa variant by denaturing electrophoresis of the spectrin tryptic digest peptides from 179 subjects: 46 controls, 78 patients with dominant (d) or non-dominant (nd) HS and 55 relatives of the patients. The confirmation of the results was obtained at the DNA level in 41 subjects. The frequency of the chromosome bearing the alpha IIa mutation was 7.6% in controls and higher (about 12-14%) in members of families with dHS as well ndHS. However, the family trees clearly showed that the mutation and the HS disease gene(s) were located on different chromosomes and inherited independently from each other. Furthermore, our study allows the conclusion that in most (if not all) cases of dHS, the alpha IIa the variant is not the cause, is not a marker, and does not influence the phenotypic expression of the disease.

An alpha-spectrin mutation responsible for hereditary elliptocytosis associated in cis with the alpha v/41 polymorphism.

The alpha 207 Leu-->Pro mutation in spectrin has recently been identified as a cause of alpha I/50-46a hereditary elliptocytosis (HE) or pyropoikilocytosis among Black people. We have found this mutation in a Moroccan family in both the heterozygous and homozygous states. The mutated alpha-spectrin allele carried, in cis, the alpha V/41 polymorphism, a common polymorphism altering the peptide maps and associated with a low-expression level. This is the first report of the cis combination of an HE mutation and the alpha V/41 polymorphism. Presumably, such a combination accounts for the very low expression of the abnormal allele in the heterozygous state.

Inherited haemolytic anaemia created by insertional inactivation of the alpha-spectrin gene.

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. By in situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the alpha-spectrin gene locus.

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.

A common type of the spectrin alpha I 46-50a-kD peptide abnormality in hereditary elliptocytosis and pyropoikilocytosis is associated with a mutation distant from the proteolytic cleavage site. Evidence for the functional importance of the triple helical model of spectrin.

We studied nine individuals from five unrelated families with alpha I/46-50a hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP), including one of the original HHP probands first reported by Zarkowsky and colleagues (1975. Br. J. Haematol. 29:537-543). Biochemical analysis of erythrocyte membrane proteins from these patients revealed, as a common abnormality, the presence of the alpha I/46-50a peptide after limited tryptic digestion of spectrin. The polymerase chain reaction was utilized to study the structure of the DNA encoding the alpha I domain of spectrin in the affected individuals. The DNA sequence of the alpha-spectrin gene encoding the region of the alpha-spectrin chain surrounding the abnormal proteolytic cleavage site was normal. We identified a point mutation causing the replacement of a highly conserved leucine residue by proline at position 207 in the alpha-spectrin chain, a site 51 residues to the amino-terminal side of the abnormal proteolytic cleavage site. Analysis of the proposed triple helical model of spectrin repeats reveals that the mutation occurs in helix 2 at a position directly opposite the abnormal proteolytic cleavage site in helix 3, making this the first report of a mutation occurring in helix 2 of a repeat in the alpha I domain of spectrin. These results add to the molecular heterogeneity of mutations associated with HE/HPP and provide further support for the proposed triple helical model of spectrin. Disruption of this proposed alpha-helical structure by helix-breaking proline substitutions may result in a functionally defective spectrin 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.