Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Protein 4.1 deficiency associated with an altered binding to the spectrin-actin complex of the red cell membrane skeleton.

Protein 4.1 has been defined as a major component of the subcortical skeleton of erythrocytes. It binds the spectrin--actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino acid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the molecular basis of heterozygous 4.1(-) hereditary elliptocytosis, associated with protein 4.1 partial deficiency, in nine related French families. cDNA sequencing revealed a single codon deletion (AAA) resulting in a lysine residue deletion within the 10-kD binding domain, 3' of Motif I. The mutated allele was designated allele 4.1 Aravis. In order to assess the functional effect of the codon deletion, recombinant 10-kD constructs were made and various binding assays were performed using spectrin, purified spectrin-actin complex, or red cell membranes. These experiments demonstrated that the deletion of the Lys residue clearly prevents the binding capacity. Similar results were obtained with a construct containing the Lys residue but lacking Motif I. These data strongly suggest that the binding site to the spectrin-actin complex must contain the Lys 447 (or 448), and therefore resides not only on Motif I but extends 3' of this essential motif.

Energy partition, scale by scale, in magnetic Archimedes Coriolis weak wave turbulence.

Magnetic Archimedes Coriolis (MAC) waves are omnipresent in several geophysical and astrophysical flows such as the solar tachocline. In the present study, we use linear spectral theory (LST) and investigate the energy partition, scale by scale, in MAC weak wave turbulence for a Boussinesq fluid. At the scale k^{-1}, the maximal frequencies of magnetic (Alfvén) waves, gravity (Archimedes) waves, and inertial (Coriolis) waves are, respectively, V_{A}k,N, and f. By using the induction potential scalar, which is a Lagrangian invariant for a diffusionless Boussinesq fluid [Salhi et al., Phys. Rev. E 85, 026301 (2012)PLEEE81539-375510.1103/PhysRevE.85.026301], we derive a dispersion relation for the three-dimensional MAC waves, generalizing previous ones including that of f-plane MHD "shallow water" waves [Schecter et al., Astrophys. J. 551, L185 (2001)AJLEEY0004-637X10.1086/320027]. A solution for the Fourier amplitude of perturbation fields (velocity, magnetic field, and density) is derived analytically considering a diffusive fluid for which both the magnetic and thermal Prandtl numbers are one. The radial spectrum of kinetic, S_{κ}(k,t), magnetic, S_{m}(k,t), and potential, S_{p}(k,t), energies is determined considering initial isotropic conditions. For magnetic Coriolis (MC) weak wave turbulence, it is shown that, at large scales such that V_{A}k/f≪1, the Alfvén ratio S_{κ}(k,t)/S_{m}(k,t) behaves like k^{-2} if the rotation axis is aligned with the magnetic field, in agreement with previous direct numerical simulations [Favier et al., Geophys. Astrophys. Fluid Dyn. (2012)] and like k^{-1} if the rotation axis is perpendicular to the magnetic field. At small scales, such that V_{A}k/f≫1, there is an equipartition of energy between magnetic and kinetic components. For magnetic Archimedes weak wave turbulence, it is demonstrated that, at large scales, such that (V_{A}k/N≪1), there is an equipartition of energy between magnetic and potential components, while at small scales (V_{A}k/N≫1), the ratio S_{p}(k,t)/S_{κ}(k,t) behaves like k^{-1} and S_{κ}(k,t)/S_{m}(k,t)=1. Also, for MAC weak wave turbulence, it is shown that, at small scales (V_{A}k/sqrt[N^{2}+f^{2}]≫1), the ratio S_{p}(k,t)/S_{κ}(t) behaves like k^{-1} and S_{κ}(k,t)/S_{m}(k,t)=1.

Delta zero-thalassemia in cis of beta Knossos-globin gene. Normal structure transient expression of the delta-globin gene.

We have previously described the first homozygous cases of Hb Knossos in an Algerian family. The Hb A2 was completely absent, ascertaining the presence of a delta zero-thalassemia determinant in cis of the beta Knossos S gene. Here, we investigate the affected delta-globin gene. The complete DNA sequence of the gene and its 5' and 3' flanking regions was determined. Only two nucleotide changes were recorded: a C----T substitution at -199 and an AT insertion at -448 upstream from the cap site. To examine the involvement of these changes in gene function, the delta-gene was subcloned in an expression vector and introduced into COS cells. Analysis of RNA derived from these cells, using an S1 protection assay and dot-blot hybridization, revealed qualitatively and quantitatively normal transcription. The loss of delta-globin gene activity in vivo may be due to the alteration of a tissue-specific control.

Hemoglobin Grange-Blanche [beta 27(B9) Ala----Val], a new variant with normal expression and increased affinity for oxygen.

Hemoglobin Grange-Blanche [beta 27(B9) Ala----Val] is a new variant found in a Portuguese family. The carriers present moderate erythrocytosis. Upon isoelectric focusing, Hb Grange-Blanche was slightly more cathodic than Hb A. beta Grange-Blanche chain migrated like the G gamma chain when submitted to electrophoresis in the presence of urea-Triton X-100. The precentage of Hb Grange-Blanche was about 50% in the heterozygous state. Oxygen affinity was increased (P50 = 22 mmHg), but heme-heme interaction was normal. An abnormal tryptic peptide (beta T3) was isolated using HPLC. Its composition allowed us to deduce unambiguously the amino acid change. The latter is the third mutation found in position 27 of the beta-chain. Because of its normal expression and its elevated affinity for oxygen, Hb Grange-Blanche contrasts with Hb Knossos [beta 27(B9) Ala----Ser], a beta-thalassemic variant with low affinity.

A missense mutation (Q279R) in the fumarylacetoacetate hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation.

BACKGROUND: Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules. RESULTS: Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. CONCLUSION: These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.

Hemoglobin J-Baltimore (beta 16(A13)Gly----Asp): interference with the assay of HbA1c.

Three independent cases of Hemoglobin J-Baltimore(beta 16(A13)Gly----Asp) were detected through the assay of HbA1c in diabetic patients. Using chromatography on Bio-Rex 70 resin, one large peak replaced the usually well resolved peaks of HbA1a + b and HbA1c. The species that overlapped the latter fractions was identified as HbJ1c. HbJ-Baltimore itself was identified using HPLC of the beta-chain tryptic peptides. This observation emphasizes the errors that hemoglobin variants may introduce in the assay of HbA1c.

Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop.

Spi-1/PU.1 oncogene is downregulated as proerythroblasts undergo terminal differentiation. Insertion of the Friend virus upstream of the Spi-1/PU.1 locus leads to the constitutive upregulation of Spi-1/PU.1, and a subsequent block in the differentiation of the affected erythroblasts. We have shown that sustained overexpression of Spi-1/PU.1 also inhibits the erythroid splicing of protein 4.1R exon 16, irrespective of chemical induction of differentiation. Here, we show a positive feedback loop that couples constitutive phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling to high expression of Spi-1/PU.1 in Friend erythroleukemia cells. Inhibition of PI3K/AKT results in Spi-1/PU.1 downregulation in a stepwise manner and induces cell differentiation. Chromatin immunoprecipitation assays further supported the positive autoregulatory effect of Spi-1/PU.1. Mutational analysis indicated that Ser41, but not Ser148, is necessary for Spi-1/PU.1-mediated repression of hemoglobin expression, whereas both Ser residues are required for Spi-1/PU.1 inhibition of the erythroid splicing event. We further show that inhibition of the erythroid transcriptional and splicing events are strictly dependent on distinct Spi-1/PU.1 phosphorylation modifications rather than Spi-1/PU.1 expression level per se. Our data further support the fact that Spi-1/PU.1 inhibits 4.1R erythroid splicing through two different pathways, and bring new insights into the extracellular signal impact triggered by erythropoietin on late erythroid regulatory program, including pre-mRNA splicing.

Chimeric probe-mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies.

Investigations throughout the last decade have established that cytoskeleton integrity ensures red cell deformability and mechanical stability, and that defects in one of the skeletal components usually result in more or less severe hemolytic anemias. Although a large number of molecular defects have been identified to date, many others still bypass fast and commonly used methods, such as SSCP and DGGE, mostly because of a subtle change in mRNA transcription level or a complex interaction leading to the loss of other components. We describe a ribonuclease protection assay based on a simultaneous quantification of two cytoskeletal transcripts, using a chimeric probe, emphasizing the value of a nonspecific bridging sequence, inserted between the two specific probe sequences. It is anticipated that this powerful and reliable procedure would be an additional tool in the methodology array used for screening cytoskeletal inherited abnormalities.

The genetic disorders of the red cell skeleton.

The genetic disorders of the red cell skeleton encompass hereditary spherocytosis, hereditary elliptocytosis and an array of ill-defined haemolytic anaemias. Protein chemistry and molecular genetics have illuminated the supramolecular arrangement of the skeleton, the sequence and three-dimensional structure of its protein components, the exon-intron organization of the corresponding genes, the complex splicing undergone by their transcripts. Basically, hereditary spherocytosis is often due to a defect of ankyrin, hereditary elliptocytosis usually results from alterations of spectrin or protein 4.1. Other conditions are related to changes in the anion transporter or protein 4.2. The heterogeneity of the genomic changes, their ultimate consequences at the protein level open windows on fundamental problems concerning alternative splicing of mRNAs and structure-function relationships in proteins.

[Interaction between various alkylxanthines and the proteins of the erythrocyte skeleton]. / Recherche d'une interaction entre certaines alkylxanthines et les protéines du squelette érythrocytaire.

We searched an interaction between (i) pentoxifylline and/or propentofylline, and (ii) the red cell membrane with special emphasis on the membrane skeleton. It appeared (i) that propentofylline has no permanent binding site on the membrane, (ii) that propentofylline and/or pentoxifylline do not detectably alter spectrin conformation (no change of spectrin dimer self-association or of spectrin limited digestion in the presence of trypsin), and (iii) that these compounds have no effect on membrane protein phosphorylation, particularly the cAMP-dependent and TPA-dependent phosphorylation of protein 4.1.

Association in cis of beta +-thalassemia and hemoglobin S.

A Moroccan woman was investigated because of a typical beta-thalassemia trait associated with a low-percentage (11%) hemoglobin (Hb) variant. The beta-thalassemia trait was manifested by a microcytosis, a high HbA2 (above 6%), and an increase of the alpha/beta biosynthetic ratio (1.31). The variant was identified to HbS by amino acid analysis of the abnormal peptide (beta T1) and by DNA mapping with Sau I (Mst II) restriction endonuclease. No additional amino acid substitution was recorded in the beta s-chain. The reduction of beta-globin synthesis occurred exclusively at the expense of the beta s-chain. These results are consistent with the existence of a beta s mutation and a beta +-thalassemia in cis.

Hemoglobin Pierre-Bénite [beta 90(F6)Glu----Asp], a new high affinity variant found in a French family.

Hemoglobin Pierre-Bénite [beta 90(F6)Glu---Asp] is a new high affinity variant (P50 = 21.5 mm Hg), with normal heme-heme interaction, found in a French family. It was difficult to detect by conventional electrophoretic methods. However, the high performance liquid chromatography profile of its tryptic peptides contained an additional peak. Amino acid analysis of the corresponding peptide and determination of its sequence allowed us to identify the mutation. No instability was found. Mutations previously recorded in position 90 of the beta-chain display a positive charge shift and a reduced affinity for oxygen, whereas Hb Pierre-Bénite shows no charge shift and increased affinity for oxygen.

The Hb F composition in a Moroccan family with beta zero-thalassaemia and Hb O-Arab.

We report on a Moroccan family in which the proposita displays a picture of beta-thalassaemia intermedia, associated with heterozygous Hb O-Arab (beta 121 Glu----Lys) and a beta zero-thalassaemia trait. Hb O-Arab was ascertained by the disappearance of the Eco RI restriction site that normally overlaps the beta-globin gene codon 121. The proposita further presents high proportions of Hb F (12.1%) and of G gamma chains (68.6%). The transmission of the proband's haemoglobin markers was analyzed (the proband's husband displaying normal haemoglobin). The beta zero-thalassaemia and O-Arab genes underwent mutual exclusion. A high Hb F (9.28%) level was found in one child, in association with the beta zero-thalassaemia trait, while another child carrying the latter trait displayed normal levels of Hb F. This situation suggests that a heterocellular HPFH determinant is involved. However, there was no means to establish whether the high Hb F proportion in the mother results solely from the beta zero-thalassaemia -Hb O-Arab association or whether an additional HPFH determinant is present. No DNA deletion was detectably associated with the high proportion of Hb F. In this family, the G gamma percentage was high whenever the beta zero-thalassaemia gene was present, regardless of total Hb F percentage. This observation is consistent with the view that the control of the G gamma percentage in the adult is linked to the beta-locus.

The association of hemoglobin Knossos and hemoglobin Lepore in an Algerian patient.

We report on a 54 years-old male patient from North-Eastern Algeria who combines two hemoglobin variants that are associated with thalassemia-like disorders: Hb Lepore and Knossos (beta 27 Ala----Ser) (1, 2). A beta-thalassemia intermedia picture gradually developed and finally required splenectomy at the age of 53. Total absence of Hb A2 indicated that the beta Knossos gene is most probably flanked with a delta(0)-thalassemia gene. No DNA deletion additional to the Lepore deletion was found. Hb F was elevated (12.3%) with 24% G gamma Hb F. In whole cells, Hb Knossos, representing 70% of total hemoglobin, displayed a decreased affinity for oxygen (P50 = 35 mm Hg), a fact presumably accounting for the relatively good tolerance of the condition.

A Dde I RFLP in exon 21 of human EL1 gene, encoding protein 4.1, detectable by SSCP.

Protein 4.1 is a major component of the junctional complex at the red cell skeleton. Genomic studies have recently evidenced that the encoding gene (EL1 locus) is present in a single copy per haploid genome. Several RFLPs have already been characterized within intron sequences. Here, we describe the first RFLP found within the coding sequence. This polymorphism (C or T at position 2723, in exon 21) does not affect the amino acid sequence (Thr-->Thr). It can be detected by either Dde I restriction digestion of an appropriate PCR product, or simply by SSCP These findings should facilitate analysis of families with 4.1 deficiencies causing hereditary elliptocytosis.

A large deletion within the protein 4.1 gene associated with a stable truncated mRNA and an unaltered tissue-specific alternative splicing.

Protein 4.1 is a major protein of the red blood cell skeleton. It binds to the membrane through its 30-kD N-terminal domain and to the spectrin-actin lattice through its 10-kD domain. We describe here the molecular basis of a heterozygous hereditary elliptocytosis (HE) associated with protein 4.1 partial deficiency. The responsible allele displayed a greater than 70-kb genomic deletion, beginning within intron 1 and ending within a 1.3-kb region upstream from exon 13. This deletion encompassed both erythroid and nonerythroid translation initiation sites. It accounts for the largest deletion known in genes encoding proteins of the red blood cell membrane. The corresponding mRNA was shortened by 1727 bases, due to the absence of exons 2 to 12. Nevertheless, this mRNA was stable. It showed a similar pattern in lymphoblastoid cells as in reticulocytes. Differential splicing of exons within the undeleted region remained regulated in a tissue-specific manner. Exons 14, 15, and 17a were absent from both reticulocyte and lymphocyte mRNAs, whereas exon 16 was present in reticulocytes but absent from lymphocytes. Thus, differential splicing on a local scale was not dependent on the overall structure of protein 4.1 mRNA in this particular instance.

Beta+-thalassemia in cis of a sickle cell gene: occurrence of a promoter mutation on a beta s chromosome.

An atypical sickle cell trait with a very low level of hemoglobin S and features of heterozygous beta-thalassemia was recently described. In vitro globin chain synthesis strongly suggested the presence of the two abnormalities on the same chromosome. We report the corresponding beta S-thal gene. DNA sequence revealed a C----T base substitution in the distal promoter element CACCC, at position-88 from the cap site, in addition to the expected GAG----GTG mutation responsible for the structural variant (beta 6 Glu----Val). Reticulocyte mRNA titration and transient assay of the mutant gene in COS cells showed a defect in beta-mRNA production. Restriction haplotype and DNA sequence analyses revealed that the doubly mutated gene is associated with haplotype 19 (or Benin/Algeria haplotype). In particular, we found the (AT)9(T)4 repeated sequences specifically encountered 5' to the beta S gene of Benin Algeria type. These results support the view that the beta S-thal gene resulted from an independent thalassemic mutation having occurred on a beta S chromosome rather than (a) from a beta S mutation having altered a beta-thalassemic gene or (b) from a recombination event between two chromosomes, each carrying one of the mutations.