Molecular defect of truncated beta-spectrin associated with hereditary elliptocytosis. Beta-spectrin Gottingen

Abstract Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

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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

Blood ◽

10.1182/blood.v80.4.1066.1066 ◽

1992 ◽

Vol 80 (4) ◽

pp. 1066-1073 ◽

Cited By ~ 15

Author(s):

M Garbarz ◽

L Boulanger ◽

S Pedroni ◽

MC Lecomte ◽

H Gautero ◽

...

Keyword(s):

Frameshift Mutation ◽

Stop Codon ◽

Molecular Defect ◽

Reading Frame ◽

Hereditary Elliptocytosis ◽

C Terminus ◽

The Family ◽

Pcr Products ◽

Polymerase Chain ◽

Self Association

Abstract 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.

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Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families

Blood ◽

10.1182/blood.v73.8.2196.bloodjournal7382196 ◽

1989 ◽

Vol 73 (8) ◽

pp. 2196-2201

Author(s):

AF Roux ◽

F Morle ◽

D Guetarni ◽

P Colonna ◽

K Sahr ◽

...

Keyword(s):

North Africa ◽

Molecular Basis ◽

Blot Hybridization ◽

Molecular Defect ◽

Dot Blot ◽

Hereditary Elliptocytosis ◽

Black Patients ◽

Allele Specific ◽

Alpha Spectrin ◽

Pcr Method

Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

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Prenatal diagnosis of hereditary elliptocytosis with molecular defect of spectrin

Prenatal Diagnosis ◽

10.1002/pd.1970070703 ◽

1987 ◽

Vol 7 (7) ◽

pp. 471-483 ◽

Cited By ~ 5

Author(s):

D. Dhermy ◽

C. Feo ◽

M. Garbarz ◽

M. C. Lecomte ◽

O. Bournier ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Molecular Defect ◽

Hereditary Elliptocytosis

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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

Blood ◽

10.1182/blood.v80.4.1066.bloodjournal8041066 ◽

1992 ◽

Vol 80 (4) ◽

pp. 1066-1073

Author(s):

M Garbarz ◽

L Boulanger ◽

S Pedroni ◽

MC Lecomte ◽

H Gautero ◽

...

Keyword(s):

Frameshift Mutation ◽

Stop Codon ◽

Molecular Defect ◽

Reading Frame ◽

Hereditary Elliptocytosis ◽

C Terminus ◽

The Family ◽

Pcr Products ◽

Polymerase Chain ◽

Self Association

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.

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Viscoelastic properties of red cell membrane in hereditary elliptocytosis

Blood ◽

10.1182/blood.v73.2.592.592 ◽

1989 ◽

Vol 73 (2) ◽

pp. 592-595 ◽

Cited By ~ 11

Author(s):

A Chabanel ◽

KL Sung ◽

J Rapiejko ◽

JT Prchal ◽

J Palek ◽

...

Keyword(s):

Viscoelastic Properties ◽

Molecular Defect ◽

Red Cell ◽

Red Cell Membrane ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Viscoelastic Parameters ◽

Rbc Membrane ◽

Dimensional Network ◽

Self Association

Abstract The viscoelastic properties of the RBC membrane are in part determined by a submembrane network of proteins consisting of spectrin alpha beta heterodimers (SpD) assembled head-to-head to form spectrin tetramers (SpT) and spectrin oligomers (SpO). SpT, in turn, are connected into a two-dimensional network by the linkage of distal ends of SpT to protein 4.1 and actin. With the micropipette technique, we determined the membrane viscoelastic properties of RBCs from a subset of patients with hereditary elliptocytosis (HE); these RBCs exhibit membrane skeletal instability, defective SpD self-association, and a molecular defect in the alpha I domain of spectrin, which is involved in the SpD-SpD contact (HE SpD alpha-SpD). The elastic modulus and viscosity of the membrane were significantly higher for the HE RBCs than for the control cells. Incubation of normal cells with N-ethyl-maleimide (NEM) produced a similar defective SpD self-association and a significant increase in the viscoelastic parameters of the membrane. The data provide evidence that the mode of assembly of membrane spectrin in the cytoskeletal protein network plays a major role in determining the rheologic behavior of erythrocyte membrane.

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Viscoelastic properties of red cell membrane in hereditary elliptocytosis

Blood ◽

10.1182/blood.v73.2.592.bloodjournal732592 ◽

1989 ◽

Vol 73 (2) ◽

pp. 592-595

Author(s):

A Chabanel ◽

KL Sung ◽

J Rapiejko ◽

JT Prchal ◽

J Palek ◽

...

Keyword(s):

Viscoelastic Properties ◽

Molecular Defect ◽

Red Cell ◽

Red Cell Membrane ◽

Protein 4.1 ◽

Hereditary Elliptocytosis ◽

Viscoelastic Parameters ◽

Rbc Membrane ◽

Dimensional Network ◽

Self Association

The viscoelastic properties of the RBC membrane are in part determined by a submembrane network of proteins consisting of spectrin alpha beta heterodimers (SpD) assembled head-to-head to form spectrin tetramers (SpT) and spectrin oligomers (SpO). SpT, in turn, are connected into a two-dimensional network by the linkage of distal ends of SpT to protein 4.1 and actin. With the micropipette technique, we determined the membrane viscoelastic properties of RBCs from a subset of patients with hereditary elliptocytosis (HE); these RBCs exhibit membrane skeletal instability, defective SpD self-association, and a molecular defect in the alpha I domain of spectrin, which is involved in the SpD-SpD contact (HE SpD alpha-SpD). The elastic modulus and viscosity of the membrane were significantly higher for the HE RBCs than for the control cells. Incubation of normal cells with N-ethyl-maleimide (NEM) produced a similar defective SpD self-association and a significant increase in the viscoelastic parameters of the membrane. The data provide evidence that the mode of assembly of membrane spectrin in the cytoskeletal protein network plays a major role in determining the rheologic behavior of erythrocyte membrane.

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