Unique alpha-spectrin mutant in a kindred with common hereditary elliptocytosis.

We report here a unique variant of alpha spectrin in a kindred with hereditary elliptocytosis. This novel red blood cell-membrane protein migrated to a position between the normal alpha- and beta-spectrin subunits in SDS polyacrylamide gel electrophoresis. It was identified as an alpha spectrin by its binding to anti-alpha spectrin antibodies, by the absence of a phosphorylation site, and by the normal 1:1 stoichiometry between total alpha- and beta-spectrin molecules. The quantity of the alpha-spectrin mutant, expressed as a percentage of the total alpha spectrin, varied from 9.9-45.2% among six affected individuals. Two-dimensional electrophoretic analysis of spectrin tryptic digests was qualitatively normal but showed a decreased quantity of a normal alpha IV fragment. The variable quantity of alpha-spectrin mutant among family members correlated directly with the increased percentage of spectrin dimers in cold low ionic strength spectrin extracts (r = 0.92) and inversely with red blood cell ghost mechanical stability (r = -0.98). The data suggest that this new alpha-spectrin mutant is responsible for decreased spectrin dimer-dimer association and for red cell instability in affected individuals.     

Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis.

Two variant spectrins have been described in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). Both are characterized by increased susceptibility of the alpha I (N-terminal) 80-kD domain to mild tryptic digestion, yielding peptides of 46-50 or 65-68 kD (T50a and T68 in our terminology). In this report we add a third unstable spectrin alpha I domain found in three kindreds with HE; alpha IT80 in this type of spectrin is cleaved by mild tryptic digestion to a 50-kD peptide (T50b) distinguished from T50a by its more basic isoelectric point. All three spectrins show impaired self-association to form oligomers. Intermediate tryptic peptides of the three unstable alpha I domains from HE spectrins were characterized by monoclonal immunoblotting and I125 limit peptide mapping and affinity purified using polyclonal anti-alpha IT80. Partial amino acid sequences of alpha I domain peptides were obtained from two unrelated patients for each of the three variant spectrins. T50a results from cleavage at arginine 250 or lysine 252 of alpha IT80; a proline replaced the normal leucine or serine at residues 254 and 255, respectively. T50b and a 19-kD peptide result from cleavage at arginine 462 or arginine 464; a proline replaced the normal residue 465 (in T19b) in one of the two patients studied. T68 results from cleavage at arginine 131. In both 68-kD peptides examined, a leucine is inserted at residue 150. The relationship of the sequence changes to the new tryptic cleavages, to the current model of alpha I domain structure, and to defective spectrin self-association is discussed.     

Tryptic digestion of spectrin in variants of hereditary elliptocytosis.

Spectrin, either in the form of unfractionated low ionic strength extracts of erythrocyte membranes or purified by chromatography on Sepharose (CL)4B, was subjected to tryptic digestion at 0 degrees C. Four patients, each with a different variant of hereditary elliptocytosis, were studied. In one patient, whose erythrocytes showed significant fragmentation on heating on 45 degrees C, such preparations generated a remarkably different pattern of polypeptide fragments on tryptic digestion at low ionic strength. In this patient 32P was released at a slower rate on tryptic digestion of labeled band 2, and an unusual 32P-labeled peptide fragment was also generated, in contrast to control preparations in which such a peptide could not be easily distinguished. There was increased susceptibility of this patient's spectrin to tryptic digestion at physiological ionic strength, but the qualitative pattern of polypeptide fragments was normal. Phosphorylation of spectrin by membrane protein kinase was markedly impaired in this patient, whereas phosphorylation of casein ws unimpaired. However, the phosphorylation of spectrin in her intact erythrocytes was normal. Our findings suggest an abnormality of spectrin structure which we postulate is causally related to the predisposition to hemolysis in this patient, but do not distinguish whether this is a primary abnormality or a post-translational modification of the spectrin molecule. The other three patients showed normal tryptic digestion of spectrin.     

A novel mobile element inserted in the alpha spectrin gene: spectrin dayton. A truncated alpha spectrin associated with hereditary elliptocytosis.

Nonviral retrotransposons, retropseudogenes, and short interspersed nuclear elements (SINEs) are mobile DNA segments capable of transposition to new genomic locations, where they may alter gene expression. De novo integration into specific genes has been described in both germ and somatic cells. We report a family with hereditary elliptocytosis and pyropoikilocytosis associated with a truncated alpha-spectrin protein. We present the biochemical characteristics of this abnormal protein and show that the alpha-spectrin gene is disrupted by a mobile element resulting in exon skipping. This element causes duplication of the insertion site and is terminated by a long poly-A tail downstream of multiple consensus polyadenylation signals. Southern blot analysis of human genomic DNA, using this element as probe, reveals one to three copies per individual. This element has no homology to any previously reported sequence and therefore appears to be a member of a novel family of mobile elements.     

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.     

Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1.

We analyzed the DNA sequence of the cDNA encoding the NH2 terminal region of beta spectrin from members of a kindred with autosomal dominant hereditary spherocytosis associated with defective protein 4.1 binding. We found a point mutation at codon 202 within the 272 amino acid NH2-terminal region of beta spectrin. TGG was changed to CGG, resulting in the replacement of tryptophan by arginine. The base change eliminates a normally occurring PvuII restriction site and creates a new MspI site. This finding enabled rapid detection or exclusion of the mutation at the DNA level among the family members, including one member for whom this analysis was performed prenatally. The mutation was found only in the affected family members and occurred as a de novo mutation in the proband. It has not been found in 20 other kindreds. The recombinant peptide derived from the normal cDNA retains the capacity to sediment with protein 4.1 and F-actin. The mutant peptide spontaneously degrades. This variant represents both the first point mutation and the first beta spectrin mutation demonstrated in autosomal dominant hereditary spherocytosis. Furthermore, the mutation is located within a conserved sequence among spectrinlike proteins and may define an amino acid critical for protein 4.1 binding activity.     

Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis.

Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.     

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.     

Interactions of spectrin in hereditary elliptocytes containing truncated spectrin beta-chains.

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.     

Abnormal oxidant sensitivity and beta-chain structure of spectrin in hereditary spherocytosis associated with defective spectrin-protein 4.1 binding.

To assess the importance of the route of glucose delivery in determining net hepatic glucose balance (NHGB) eight conscious overnight-fasted dogs were given glucose via the portal or a peripheral vein. NHGB was measured using the arteriovenous difference technique during a control and two 90-min glucose infusion periods. The sequence of infusions was randomized. Insulin and glucagon were held at constant basal levels using somatostatin and intraportal insulin and glucagon infusions during the control, portal, and peripheral glucose infusion periods (7 +/- 1, 7 +/- 1, 7 +/- 1 microU/ml; 100 +/- 3, 101 +/- 6, 101 +/- 3 pg/ml, respectively). In the three periods the hepatic blood flow, glucose infusion rate, arterial glucose level, hepatic glucose load, arterial-portal glucose difference and NHGB were 37 +/- 1, 34 +/- 1, 32 +/- 3 ml/kg per min; 0 +/- 0, 4.51 +/- 0.57, 4.23 +/- 0.34 mg/kg per min; 101 +/- 5, 200 +/- 15, 217 +/- 13 mg/dl; 28.5 +/- 3.5, 57.2 +/- 6.7, 54.0 +/- 6.4 mg/kg per min; +2 +/- 1, -22 +/- 3, +4 +/- 1 mg/dl; and 2.22 +/- 0.28, -1.41 +/- 0.31, and 0.08 +/- 0.23 mg/kg per min, respectively. Thus when glucose was delivered via a peripheral vein the liver did not take up glucose but when a similar glucose load was delivered intraportally the liver took up 32% (P less than 0.01) of it. In conclusion portal glucose delivery provides a signal important for the normal hepatic-peripheral distribution of a glucose load.     

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.     

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.     

Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis.

We describe a spectrin variant characterized by a truncated beta chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderate hemolytic anemia with striking spherocytosis and mild spiculation of the red cells. We describe the biochemical characteristics of this truncated protein which constitutes only 10% of the total beta spectrin present on the membrane, resulting in spectrin deficiency. Analysis of reticulocyte cDNA revealed the deletion of exons 22 and 23. We show, using Southern blot analysis, that this truncation results from a 4.6-kb genomic deletion. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we show that (a) the mutated gene is efficiently transcribed and its mRNA abundant in reticulocytes, (b) the mutant protein is normally synthesized in erythroid progenitor cells, (c) the stability of the mutant protein in the cytoplasm of erythroblasts parallels that of the normal beta spectrin, and (d) the abnormal protein is inefficiently incorporated into the membrane of erythroblasts. We conclude that the truncation within the beta spectrin leads to inefficient incorporation of the mutant protein into the skeleton despite its normal synthesis and stability. We postulate that this misincorporation results from conformational changes of the beta spectrin subunit affecting the binding of the abnormal heterodimer to ankyrin, and we provide evidence based on binding assays of recombinant synthetic peptides to inside-out-vesicles to support this model.     

Severe hyperbilirubinemia in a neonate with hereditary spherocytosis due to a de novo ankyrin mutation: A case report

BACKGROUNDHereditary spherocytosis (HS) is a common type of hemolytic anemia caused by a red cell membrane disorder. HS type 1 (HS1) is mostly caused by mutations in ankyrin (ANK1). Newborns with HS1 usually only exhibit anemia and mild jaundice. We herein report a case of HS1 and discuss its clinical characteristics.CASE SUMMARYA 2-d-old male full-term newborn was admitted to our hospital with severe, intractable neonatal jaundice. Laboratory investigations showed hemolytic anemia and hyperbilirubinemia and excluded immune-mediated hemolysis. The patient underwent two exchange transfusions and one plasmapheresis resulting in significantly reduced serum bilirubin. Hematologic analyses and genomic DNA sequencing studies were performed. The trio clinical exome sequencing revealed a de novo null heterozygous mutation in the patient''s ANK1 gene: c.841C > T(p.Arg281Ter). This mutation results in the premature termination of the ANK1 protein.CONCLUSIONOur case demonstrates that genetic analysis can be an essential method for diagnosing HS when a newborn has severe hyperbilirubinemia.     

Isoelectric focusing of spectrin components in hereditary spherocytosis.

1. By isoelectric focusing in 8 M urea, spectrin purified from normal human erythrocytes was resolved into 12 to 15 peptide bands differing by their isoelectric point. Most of them were focused between pH 6.4 and 5.2, one at pH 8.7 and some minor components between pH 7.4 and 6.8. 2. The results were not influenced by the erythrocyte population age. 3. Spectrin purified from erythrocytes of five patients with hereditary spherocytosis gave similar isoelectric focusing patterns, with the exception of the lack of the pH 8.7-focused component in two related patients.     

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.     

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.     

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.     

Clinical and molecular genetic analysis of a Chinese family with hereditary elliptocytosis caused by a novel mutation in the EPB41 gene

BackgroundHereditary elliptocytosis (HE) is a heterogeneous red blood cell membrane disorder characterized by the presence of elliptocytes on a peripheral blood smear. Clinical manifestations of HE vary widely from asymptomatic carriers to patients with severe transfusion‐dependent anemia. Most patients are asymptomatic or have mild anemia, which hinders diagnosis. The proband in this case had mild anemia and jaundice over a period of 4 years, the etiology of which was unclear. Hence, he was admitted to our hospital for further diagnosis.MethodsPeripheral blood smears and routine blood tests were performed and biochemical parameters of the proband, and his family members were determined. To confirm the diagnosis, gene mutations were screened in the proband using next‐generation sequencing (NGS) and verified by Sanger sequencing in other family members.ResultsA novel mutation (c.1294delA, p.Ser432 fs) in exon 15 of the EPB41 gene was detected in the proband and his family members. This mutation results in a frameshift and a premature stop codon at position 455, encoding a truncated protein. The variant was likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. SWISS‐MODEL protein structure prediction indicated partial loss of the spectrin and actin binding and C‐terminal domains.ConclusionA heterozygous mutation 1294delA in exon 15 of the EPB41 gene was identified using NGS and Sanger sequencing in members of a Chinese family. This identification expands the spectrum of EPB41 mutations and contributes to the genetic diagnosis of families with HE.     

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.