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J Delaunay

Publications and source records attributed to J Delaunay.

At least 55 records · Page 3Linked to original sources
PubMed1997

A markedly disrupted skeletal network with abnormally distributed intramembrane particles in complete protein 4.1-deficient red blood cells (allele 4.1 Madrid): implications regarding a critical role of protein 4.1 in maintenance of the integrity of the red blood cell membrane.

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG --> AGG) of the protein 4.1 gene [the 4.1 (-) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 +/- 63/microm2; normal, 504 +/- 36/microm2). The number of basic units in the skeletal network was strikingly reduced (131 +/- 21/microm2; normal, 548 +/- 39/microm2), with decreased small-sized units (17 +/- 4/microm2; normal, 384 +/- 52/microm2) and increased large-sized units (64% +/- 14%; normal, 5% +/- 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.

Anemia, Hemolytic↗
PubMed1997

Modulation of clinical expression and band 3 deficiency in hereditary spherocytosis.

We present two novel alleles of the anion-exchanger 1 (AE1) gene, allele Coimbra and allele Mondego. Allele Coimbra (V488M, GTG --> ATG) affects a conserved position in the putative second ectoplasmic loop of erythrocyte band 3. In 15 simple heterozygotes, it yielded a mild form of hereditary spherocytosis (HS) with band 3 deficiency (-20% +/- 2%) and a reduced number of 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonate (H2DIDS) binding sites (-35%). However, two additional heterozygotes presented with an aggravated HS and a more pronounced reduction of band 3 (-40%) and of H2DIDS binding sites (-48%). They carried, in trans to allele Coimbra, allele Mondego, defined by two mutations: E40K, GAG --> AAG, the known mutation Montefiore, and P147S, CCT --> TCT, a novel mutation, both located in the cytoplasmic domain of band 3. Allele Mondego itself resulted in no clinical or hematologic HS signs in the simple heterozygous state. Yet it yielded a slight decrease in band 3 (-6% to -12%) and in the number of H2DIDS binding sites (-19%). Thus, the more pronounced decrease in band 3 in the two compound heterozygotes derived from the additive effects of two unequally expressed AE1 alleles, resulting in a more severe clinical picture.

Alleles↗
PubMed1997

Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA.

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3'-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3' UTR, over 3 kb, accounts for one-half of the mature mRNA.

Actins↗
PubMed1997

Ankyrin Bugey: a de novo deletional frameshift variant in exon 6 of the ankyrin gene associated with spherocytosis.

We describe a case of spherocytosis in a French child splenectomized at age 10 years. The parents were devoid of any clinical, hematological, or biochemical abnormalities. Following splenectomy, the proposita exhibited a reduction of red cell membrane ankyrin. The variable number of dinucleotide repeats associated with the erythroid ankyrin gene (ANK1) were studied at the genomic level. The father, the mother, and the proposita had the AC14/AC11, AC14/AC14, and AC14/AC11 genotypes, respectively, although the proposita exhibited a pattern consistent with an AC14,-combination at the cDNA level. We thought there could be a de novo mutation in the ANK1 allele of paternal origin (AC11). A false paternity seemed most unlikely. Based on PCR-amplification of exons, SSCP analysis, and, when appropriate, nucleotide sequencing, we found a one-nucleotide deletion in codon 146 (exon 6): 521delC, ACG-->AG. This placed in phase a TAG triplet normally overlapping codons 150 and 151. Early interruption of translation presumably accounted for the premature degradation of mutant mRNA. Restriction analysis confirmed the presence of the mutation in the proposita and its absence in the parents. The variant was designated ankyrin Bugey.

Ankyrins↗
PubMed1997

A variant of the EPB3 gene of the anti-Lepore type in hereditary spherocytosis.

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS-PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in-frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt -7 to -3), and the other within exon 13 (nt 1494-1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences. At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478-499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.

Adolescent↗
PubMed1997

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis.

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG-->GAG, and P854L, CCG-->CTG) and, additionally, the mutation: G714R, GGG-->AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA-->AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.

Amino Acid Substitution↗
PubMed1997

Novel band 3 variants (bands 3 Foggia, Napoli I and Napoli II) associated with hereditary spherocytosis and band 3 deficiency: status of the D38A polymorphism within the EPB3 locus.

We report three novel variants of band 3 associated with hereditary spherocytosis: band 3 Foggia (311delC; ACCCAC-->ACCAC), band 3 Napoli I (447insT; TCT-->TTCT) and band 3 Napoli II (1783N; ATC-->AAC). The first two mutations resulted in premature termination of translation, making one haploid set of band 3 mRNA unavailable. Since it affected a highly conserved position at the terminal end of transmembrane domain 11, the third mutation prevented one haploid set of band 3 from becoming incorporated or stabilized into the membrane. These three mutations resulted in a reduction of the band 3 level in the red cell membrane (by 20-25%) and were dominantly transmitted. The D38A substitution (GAC-->GCC) is a low frequency change of band 3. In one compound heterozygote D38A/Napoli II, a markedly aggravated picture required early splenectomy. In contrast, the D38A change was not associated with deterioration in another compound heterozygote, carrying in trans, the previously recorded R760W mutation (CGG-->TGG). In the aggravated case, SSCP analysis did not exhibit any additional change in the two EPB3 alleles. Nor did it show any alteration in the exons of the two ANK1 alleles, and the aggravating factor remained elusive. The D38A alteration should be regarded as an innocuous polymorphism.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1997

Frequent de novo mutations of the ANK1 gene mimic a recessive mode of transmission in hereditary spherocytosis: three new ANK1 variants: ankyrins Bari, Napoli II and Anzio.

A subset of spherocytosis cases associated with mutations of the ANK1 gene present an apparently recessive inheritance pattern on a clinical and haematological basis. We identified three novel out-of-frame deletions in the ANK1 gene: allele Bari (1361delG), Napoli II (2883delC) and Anzio (3032delCA) in three Italian patients, two of whom have been splenectomized. Analysis of the cDNA showed small or trace amounts of ankyrin mRNAs in Bari, Napoli II and Anzio. The parents were normal clinically and haematologically and did not carry the mutations exhibited by their children. We confirmed the de novo character of the HS mutations based on paternity testing. Recessive HS associated with the ANK1 gene is probably rarer than initially thought, and spherocytosis may often be due to de novo mutations.

Ankyrins↗
PubMed1997

Localization of the congenital dyserythropoietic anemia II locus to chromosome 20q11.2 by genomewide search.

Congenital dyserythropoietic anemias (CDA) are genetic disorders characterized by anemia and ineffective erythropoiesis. Three main types of CDA have been distinguished: CDA I and CDA III, whose loci have been already mapped, and CDA II (MIM 224100), the most frequent among CDAs, which is transmitted as an autosomal recessive trait and is known also as "HEMPAS" (hereditary erythroblast multinuclearity with positive acidified serum). We have recruited a panel of well-characterized CDA II families and have used them to search for the CDA II gene by linkage analysis. After the exclusion of three candidate genes, we ob-tained conclusive evidence for linkage of CDA II to microsatellite markers on the long arm of chromosome 20 (20q11.2). A maximum two-point LOD score of 5.4 at a recombination fraction of .00 was obtained with marker D20S863. Strong evidence of allelic association with the disease was detected with the same marker. Some recombinational events established a maximum candidate interval of approximately 5 cM.

Anemia, Dyserythropoietic, Congenital↗
PubMed1996

Hereditary spherocytosis with band 3 deficiency. Association with a nonsense mutation of the band 3 gene (allele Lyon), and aggravation by a low-expression allele occurring in trans (allele Genas).

We describe an 18-year-old with moderate hereditary spherocytosis. The condition was associated with a 35% decrease in band 3. The underlying mutation was Arg to stop at codon 150 (CGA-->TGA) and was designated R150X, which defined allele Lyon of the EPB3 gene. The inheritance pattern was dominant. However, the mother, who also carried the allele Lyon, had a milder clinical presentation and only a 16% decrease of band 3. We suggested that the father had transmitted a modifying mutation that remained silent in the heterozygous state. Nucleotide sequencing after single strand conformation polymorphism analysis of the band 3 cDNA and promoter region revealed a G-->A substitution at position 89 from the cap site in the 5'-untranslated region, designated 89G-->A, which defined allele Genas. A ribonuclease protection assay showed that (1) the allele Genas (father) resulted in a 33% decrease in the amount of band 3 mRNA, (2) the reduction caused by the allele Lyon (mother) was 42%, and (3) the compound heterozygous state for both alleles (proband) resulted in a 58% decrease. These results suggest that some mildly deleterious alleles of the EPB3 gene are compensated for by the normal allele in the heterozygous state. They are shown through the aggravation of the clinical picture, based on more obvious molecular alterations when they occur in trans to an allele causing a manifest reduction of band 3 membrane protein concentration.

Alleles↗
PubMed1996

Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation.

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5' end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.

Alternative Splicing↗
PubMed1996

Hemoglobin Debrousse (beta 96[FG3]Leu-->Pro): a new unstable hemoglobin with twofold increased oxygen affinity.

Hemoglobin Debrousse (beta 96[FG3]Leu-->Pro) is a new unstable variant, with high oxygen affinity responsible, in the steady state, for an apparently well-compensated chronic hemolytic anemia. The functional properties of this variant are due to the replacement of a leucine residue which is involved in the hydrophobic environment of the proximal side of the heme. This electrophoretically neutral hemoglobin was found as a de novo case in a 6-year-old girl suffering from severe anemia with hemolysis and transient aplastic crisis, following infection by parvovirus B19.

Adult↗
PubMed1996

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.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Spectrin mutations in hereditary elliptocytosis and hereditary spherocytosis.

Hereditary elliptocytosis (HE), its aggravated form hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS) designate a set of congenital hemolytic syndromes. The responsible mutations lie in several genes encoding proteins of the red cell membrane. In particular, they involve the SPTA1 and SPTB genes that encode erythroid spectrin alpha- and beta-chains, respectively. In situ, spectrin is a alpha 2 beta 2 fibrillar tetramer resulting from the head-to-head self-association of two alpha beta dimers. In HE, the 24 known alpha-chain mutations lie in the self-association site or its vicinity, whereas the 17 beta-chain mutations occur in the self-association site itself (record of November 30, 1995). Allele alpha LELY (LELY: Low Expression LYon) is found in ethnic groups remote from one another with a uniform frequency (20-30% of all alpha-alleles). It allows an expanded expression of any HE alpha-allele located in trans and results in severe HE or in HPP. In HS, a number of spectrin mutations have been recorded recently. Allele alpha LEPRA (LEPRA: Low Expression PRAgue) would occur in a recurrent fashion.

Elliptocytosis, Hereditary↗
PubMed1996

Ankyrin Napoli: a de novo deletional frameshift mutation in exon 16 of ankyrin gene (ANK1) associated with spherocytosis.

We report a case of apparently recessive hereditary spherocytosis in an Italian child. The proband exhibited a reduction of overall ankyrin in the red cell membrane. The parents were free of any haematological manifestations. The VNDR associated with the ankyrin gene (ANK1) were consistent with the following diplotypes: AC11/ AC14 (father), AC14/AC14 (mother) and AC11/AC14 (child). The cDNA of the patient disclosed the expression of the AC11 allele only. As a consequence, we put forward the hypothesis of a de novo inactivation affecting the ankyrin allele of maternal origin (AC14) and accounting for the disease. PCR amplification of exons, SSCP analysis and nucleotide sequencing disclosed a polymorphism: GAC --> AAC; Asp --> Asn in codon 328 of exon 10, and a one-nucleotide deletion : CTG --> CG in codon 573 of the exon 16. This frameshift mutation placed in phase the TGA triplet that normally overlaps codons 636 and 637. Termination of translation near the middle of ankyrin mRNA coding sequence resulted, presumably, in its premature degadation. The present allele has been designated allele Napoli.

Amino Acid Sequence↗
PubMed1996

Hb Aubenas [beta 62(B8)Glu-->Gly]: a new variant normally synthesized, affecting the same codon as in Hb E.

Hb Aubenas [beta 26(B8)Glu-->Gly] is a mildly unstable variant that was found in a French family without hematological or clinical features. The structural abnormality was determined by protein chemistry methods, including tandem mass spectrometry, and was confirmed by Apa I digestion of a polymerase chain reaction-amplified DNA fragment. Although the substitution involves the same residue as in Hb E, the new nucleotide sequence does not create an additional out-of-frame splice site. The mutated chain is therefore normally synthesized.

Adolescent↗