Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid-->lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Abstract Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.

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Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid-->lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Blood ◽

10.1182/blood.v81.8.2155.bloodjournal8182155 ◽

1993 ◽

Vol 81 (8) ◽

pp. 2155-2165 ◽

Cited By ~ 3

Author(s):

AC Rybicki ◽

JJ Qiu ◽

S Musto ◽

NL Rosen ◽

RL Nagel ◽

...

Keyword(s):

Sequence Analysis ◽

Hemolytic Anemia ◽

Cytoplasmic Domain ◽

Band 3 ◽

Structural Abnormality ◽

Cell Deformability ◽

Whole Cell ◽

Rbc Membrane ◽

Protein 4.2 ◽

Lysine Substitution

Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.

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Band 3 Tuscaloosa: Pro327----Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2

Blood ◽

10.1182/blood.v80.2.523.bloodjournal802523 ◽

1992 ◽

Vol 80 (2) ◽

pp. 523-529 ◽

Cited By ~ 2

Author(s):

P Jarolim ◽

J Palek ◽

HL Rubin ◽

JT Prchal ◽

C Korsgren ◽

...

Keyword(s):

Hemolytic Anemia ◽

Binding Capacity ◽

Cytoplasmic Domain ◽

Band 3 ◽

Molecular Defect ◽

Band 3 Protein ◽

Additional Line ◽

Peripheral Proteins ◽

Protein 4.2 ◽

Partial Deficiency

Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56--- -Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic.

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Band 3 Tuscaloosa: Pro327----Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2

Blood ◽

10.1182/blood.v80.2.523.523 ◽

1992 ◽

Vol 80 (2) ◽

pp. 523-529 ◽

Cited By ~ 58

Author(s):

P Jarolim ◽

J Palek ◽

HL Rubin ◽

JT Prchal ◽

C Korsgren ◽

...

Keyword(s):

Hemolytic Anemia ◽

Binding Capacity ◽

Cytoplasmic Domain ◽

Band 3 ◽

Molecular Defect ◽

Band 3 Protein ◽

Additional Line ◽

Peripheral Proteins ◽

Protein 4.2 ◽

Partial Deficiency

Abstract Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56--- -Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic.

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Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of α-Spectrin.

Blood ◽

10.1182/blood.v106.11.1662.1662 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1662-1662

Author(s):

Raymond F. Robledo ◽

Amy J. Lambert ◽

Babette Gwynn ◽

Lucy B. Rowe ◽

Diana M. Gilligan ◽

...

Keyword(s):

Hemolytic Anemia ◽

Membrane Skeleton ◽

Band 3 ◽

Chromosome 1 ◽

Major Protein ◽

Blood Profile ◽

Rbc Membrane ◽

C Terminus ◽

Sds Page ◽

Rbc Membrane Skeleton

Abstract The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

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Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia.

Blood ◽

10.1182/blood.v110.11.1728.1728 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1728-1728

Author(s):

Jason M. Wooden ◽

Greg L. Finney ◽

Michael J. MacCoss ◽

Luanne L. Peters ◽

Diana M. Gilligan

Keyword(s):

Mouse Models ◽

Hemolytic Anemia ◽

Membrane Skeleton ◽

Tandem Mass ◽

Secondary Effects ◽

Knock Out ◽

Rbc Membrane ◽

Protein 4.2 ◽

Knock Out Mice ◽

Rbc Membrane Skeleton

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up’ analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.

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A New Case of KLF1 G973A Mutation and Congenital Dyserythropoeitic Anemia (CDA)- Further Definition of Emerging New Syndrome and Possible Association with Gonadal Dysgenesis

Blood ◽

10.1182/blood.v118.21.2101.2101 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2101-2101 ◽

Cited By ~ 3

Author(s):

Yaddanapudi Ravindranath ◽

Gerald Goyette ◽

Steven Buck ◽

Manisha Gadgeel ◽

Alan Dombkowski ◽

...

Keyword(s):

Sequence Analysis ◽

Nucleotide Sequence ◽

Hemolytic Anemia ◽

Gonadal Dysgenesis ◽

Band 3 ◽

Dominant Negative Effect ◽

Nucleotide Sequence Analysis ◽

Severe Anemia ◽

Fetal Hydrops ◽

Male Sex

Abstract Abstract 2101 Arnaud et ala recently reported on two children with an as yet unclassified CDA, characterized by hemolytic anemia with high fetal hemoglobin (HbF), presence of embryonic hemoglobin Portland (HbPort) and absence on red blood cells (RBC) of CD44, an adhesion molecule, aquaporin 1 (AQP1) and the linked In(Lu) and Colton blood groups. The underlying molecular defect involves the erythroid transcription factor KLF1 with a mutation in zinc finger 2 at position 973 of Guanine to Adenine. The G973A mutation results in substitution of glutamate to lysine at position 325 in the transcribed protein. The CDA results from a dominant negative effect of KLF1 E325K on the gamma-globin repressor BCL11A, leading to the persistence of fetal erythropoiesis, high Hb F and HbPort. The absence of CD44, AQP1 and deficiency of certain cytoskeletal proteins in the RBC indicate that KLF1 is necessary for the terminal differentiation of RBC. The presence of CD44 on the leucocytes in these cases would appear to suggest an erythroid restrictive effect of the mutation. Of note, the child in whom the E325K mutation was first identified, a 46XY male, was described to have micropenis and hypospadias; the hemolytic anemia was severe and required splenectomy. The original case was that of phenotypic female with 46XX and the clinical course was much milder. We have identified a new case (a caucasian child) with the KLF1 E325K mutation. Comparison with the clinical findings in the two published cases indicates that KLF1 E325K mutation may cause profound developmental disturbances in organs beyond erythroid development, particularly of the gonads. Our patient, now 5 yrs old, was followed with an undiagnosed hemolytic anemia, which started with fetal hydrops at 23 weeks gestation, requiring intrauterine transfusions. Because of the presence of spherocytes on the blood smear and history of renal stones, a mutant band 3 was suspected; however direct nucleotide sequence analysis of the promoter and coding region exons of the band 3 gene from amplified genomic DNA of the proband was normal. Splenectomy was done at 4-years-of-age and the emerging picture was one of extreme normoblastemia, with the presence of both spherocytes and massively enlarged erythrocytes. Osmotic gradient ektacytometry showed reduced erythrocyte deformability with a population of swollen erythrocytes. Hemoglobin analyses revealed a HbF of31% and, HgPort (isoelectric focusing done by James Hoyer at Mayo Laboratories). Flow cytometry demonstrated that CD44 was not expressed on patient's erythrocytes but was expressed on lymphocytes. AQP1 was absent on western blots of RBC ghosts. Nucleotide sequence analysis of the KLF1 gene identified the KLF1 E325K mutation in lymphocyte DNA from the patient, but not in her parents. In addition to the severe anemia, the child who is 46XY, has female external genitalia. Ultrasonography demonstrated a vagina and uterus. Surgically excised streak gonads showed no cellular elements. Array comparative genomic hybridization studies did not reveal any copy number variations in male sex determining genes, but identified polymorphic deletion of estrogen metabolizing UGT2B17. Together these 3 cases suggest that KLF1 E325K may cause more severe anemia and gonadal dysgenesis in 46XY fetuses while the disease is considerably milder in 46XX individuals. Both of the 46 XY cases also had fetal hydrops and were salvaged with transfusions in utero, suggesting that KLF1 mutations may cause early loss of male fetuses. Given that KFL1 interacts with more than 1000 proteins,b,c it is likely that KLF1 is important in the developmental regulation of non erythroid genes including male sex determining genes. Disclosures: No relevant conflicts of interest to declare.

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Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

Blood ◽

10.1182/blood-2009-07-232199 ◽

2010 ◽

Vol 115 (9) ◽

pp. 1804-1814 ◽

Cited By ~ 10

Author(s):

Raymond F. Robledo ◽

Amy J. Lambert ◽

Connie S. Birkenmeier ◽

Marius V. Cirlan ◽

Andreea Flavia M. Cirlan ◽

...

Keyword(s):

Missense Mutation ◽

Hemolytic Anemia ◽

Stop Codon ◽

Premature Stop Codon ◽

Band 3 ◽

Cysteine Residue ◽

Normal Band ◽

Junctional Complex ◽

Rbc Membrane ◽

C Terminus

AbstractFive spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

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Faculty Opinions recommendation of An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1090559.543838 ◽

2007 ◽

Author(s):

Achille Iolascon

Keyword(s):

Amino Acid ◽

Cytoplasmic Domain ◽

Band 3 ◽

Hairpin Loop ◽

Beta Hairpin

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Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis

Blood ◽

10.1182/blood.v70.3.766.bloodjournal703766 ◽

1987 ◽

Vol 70 (3) ◽

pp. 766-772

Author(s):

T Coetzer ◽

J Lawler ◽

JT Prchal ◽

J Palek

Keyword(s):

Hemolytic Anemia ◽

Genetic Defect ◽

Clinical Severity ◽

Lower Percentage ◽

Structural Abnormality ◽

Clinical Expression ◽

Related Disorder ◽

Asymptomatic Carriers ◽

Hereditary Elliptocytosis ◽

Self Association

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.

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