Red blood cell membrane disorders

The red blood cell membrane is a multi-component structure that is responsible for many of the physiological functions and mechanical properties of the cell. Defects in any of these components can manifest as clinical disorders involving the erythrocytes. In the past few years there have been major advances in our understanding of the molecular basis of these disorders, in which mutational analysis has clarified many questions about the structure–function relationship of components of the red cell membrane. Some of these recent findings will be the subject of this review. A detailed discussion of the structure of the red cell membrane and the pathophysiology and clinical aspects of its disorders can be found in several recent reviews (Lux Delaunay, 1995; Hassoun Gallagher et al, 1998). An updated catalogue of mutations causing red cell membrane disorders is available at the website http://www.kidscancer.net/membrane/. The red cell membrane comprises a lipid bilayer, integral membrane proteins and a membrane skeleton (Fig 1). The red cell membrane skeleton is a multi-protein complex formed by structural proteins including α and β spectrin, ankyrin, protein 4.1 and actin. The membrane skeleton proteins interact with the lipid bilayer and transmembrane proteins to give the red cell membrane its strength and integrity. They interact with each other to form a scaffolding on the inner surface of the lipid bilayer. α and β spectrin interact side-to-side to form flexible rod-like heterodimers which self-associate head-to-head to form tetramers. The tetramers are linked by ankyrin to the cytoplasmic domain of the integral membrane protein band 3. Protein 4.2 binds to band 3 at the same position and may enhance the ankyrin–band 3 interaction. Multiple spectrin tetramers interact at their tail ends and with actin protofilaments, tropomyosin, tropomodulin and adducin to form junctional complexes. Protein 4.1, which also binds to the integral membrane protein glycophorin C, interacts with β spectrin at the actin-binding domain and increases the affinity of the spectrin–actin binding. . Schematic model of the red cell membrane, with the vertical and horizontal interaction of its components indicated. Estimated frequencies of mutations in different membrane proteins in HS and HE/HPP are as follows. Vertical interaction: hereditary spherocytosis: band 3, ~20%; protein 4.2, ~5%; ankyrin, ~45%; β spectrin, ~30%. Horizontal interaction: hereditary elliptocytosis/hereditary pyropoikilocytosis: β spectrin, ~5%; α spectrin, ~80%; protein 4.1, ~15%. The relative position of the various proteins is correct, but the proteins and lipids are not drawn to scale. Adapted from Lux Palek, 1985), defects in horizontal interactions result in hereditary elliptocytosis or hereditary pyropoikilocytosis, and defects in vertical interactions lead to hereditary spherocytosis. Recent identification of the many defects that underlie red cell membrane disorders has shown that this model is essentially correct. Hereditary spherocytosis (HS) is the most common cause of non-immune haemolytic anaemia in people of Northern European ancestry, with a prevalence of approximately 1 in 2000. In about 75% of the cases the inheritance follows an autosomal dominant pattern, and in about 25% the occurrence of the disease is sporadic in nature (Agre et al, 1986; Eber et al, 1990). About half of the sporadic cases are probably caused by a recessive form of HS and the rest by spontaneous new mutations (Eber et al, 1990; Miraglia del Giudice et al, 1998a, b). Clinically, HS is characterized by the presence of spherocytes in peripheral smears with varying degrees of haemolysis and splenomegaly. There is increased fragility of the red cell membrane, leading to vesiculation of the membrane, loss of membrane surface area, and trapping and destruction of the red cells in the spleen. Splenectomy ameliorates the degree of haemolysis and is indicated in severely affected patients, but the intrinsic abnormality of the red cells remains after the procedure. Early biochemical analysis of the membrane skeleton proteins showed that spectrin is deficient in patients with HS (Agre et al, 1985). The severity of the disease and response to splenectomy correlates with the amount of spectrin deficiency (Agre et al, 1986; Eber et al, 1990). Cytogenetic and genetic linkage analyses later showed that ankyrin defects are an underlying cause of HS in some families (Lux et al, 1990; Costa et al, 1990). Subsequent work showed that most patients with HS have combined spectrin and ankyrin deficiency (Pekrun et al, 1993; Savvides et al, 1993). Our current understanding is that HS is caused by defects in the proteins involved in the vertical interactions between the membrane skeleton and the lipid bilayer. Several surveys using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis have shown that 30–45% of HS patients have combined ankyrin and spectrin deficiency, about 30% have isolated spectrin deficiency, and about 20% have band 3 deficiency (Miraglia del Giudice et al, 1994; Jarolim et al, 1996a; Dhermy et al, 1997; Lanciotti et al, 1997). Analysis by SDS-PAGE may, however, underestimate the degree of ankyrin deficiency in these patients. Quantitative analyses by radioimmunoassay and ELISA show that most HS patients have a roughly equivalent deficiency of both spectrin and ankyrin (Pekrun et al, 1993; Savvides et al, 1993). A high reticulocyte count may also mask the detection of ankyrin deficiency (Miraglia del Giudice et al, 1997a). Isolated protein 4.2 deficiency is found in a small number of American and European patients but is more common in Japan (Inoue et al, 1994). Using a dinucleotide repeat polymorphism of the ankyrin cDNA as a marker to distinguish between the two ankyrin alleles, it was shown that one ankyrin allele has reduced expression in a third of HS patients with combined spectrin and ankyrin deficiency (Jarolim et al, 1996b). This may be caused by reduced transcription of the gene or decreased stability of its transcripts. By the same method, it has been shown that de novo mutations in one of the ankyrin alleles leading to decreased expression are frequent in HS children with normal parents (Miraglia del Giudice et al, 1998a, c). Molecular analyses confirm that ankyrin defects are present in roughly half of patients with HS (Eber et al, 1996). Null mutations predominate in dominant HS. These are nonsense or frameshift mutations that result in either unstable ankyrin mRNA transcripts or truncated peptides. Mutant ankyrins Bari, Bugey, Duisburg, Einbeck, Marburg, Napoli I, Osterholz and Stuttgart are truncated in the band 3 binding domain (Eber et al, 1996; Miraglia del Giudice et al, 1996; Morléet al, 1997; Randon et al, 1997), ankyrins Anzio, Napoli II and Porta Westfalica are affected in the spectrin binding domain, and ankyrins Bovenden and Saint-Etienne 1 and 2 have premature termination within the regulatory domain (Eber et al, 1996; Hayette et al, 1998). In ankyrin Rakovnik a nonsense mutation within the regulatory domain leads to a selective deficiency of the major ankyrin isoform, protein 2.1 (Jarolim et al, 1995b). A point mutation in position −204 of the promoter region in a with HS leads to decreased of ankyrin (Eber et al, 1996). The clinical with these mutations from haemolysis to This may be by different of for the either from of the normal ankyrin allele or ankyrin Several ankyrin defects have been found in patients with recessive HS. the mutations found in dominant these are or promoter A point mutation in the ankyrin promoter the is It was found in of families with recessive HS in one (Eber et al, 1996). In two of these families a mutation in the other ankyrin allele was ankyrins and Ankyrin was present in a red cells more deficient in band 3 than in spectrin or ankyrin, which is to the in other ankyrin It from a mutation in the band 3 binding domain and a decreased affinity for band 3. Ankyrin was found in with recessive HS and a mutation in a that may result in of the HS patients with ankyrin defects have spherocytosis other and anaemia from to (Eber et al, 1996; Miraglia del Giudice et al, al, 1997; Randon et al, 1997; Hayette et al, 1998). In patients with dominant defects are affected than with recessive however, there is spectrin deficiency has been with it is that mutations in have been as a cause of the α spectrin is in in red cells and β spectrin is the in of the membrane skeleton et al, 1998). Several mutations in β spectrin have been in HS. In spectrin a point mutation the of β spectrin, to the protein 4.1 binding site et al, binding to protein 4.1, the spectrin unstable and et al, and leads to spectrin deficiency and dominant HS. In and point mutations the site of the mutation et al, 1997). In one of recessive HS the has a point mutation in position of the β spectrin which an to a et al, 1997). The mutation in the allele has not been Clinically, HS from to in patients with defects et al, 1993; Hassoun et al, 1997; et al, Miraglia del Giudice et al, is and is with a of or that are not in other forms of HS. α spectrin is to in of that in the red cell membrane et al, 1997). A with a haemolytic anaemia and of spherocytosis was found to have an α spectrin allele a truncated spectrin and a α spectrin spectrin allele et al, 1996). The allele a mutation that premature termination of and a in α spectrin This allele is in linkage with an α spectrin or spectrin that is found in families with HS et al, 1993; et al, 1997). In these patients, analysis of their membrane spectrin the but analysis the presence of both the allele and a allele the polymorphism et al, 1997). These that the allele in to the the allele is in these patients. of the α spectrin allele with α spectrin allele a mutation may be a common cause of recessive HS. Band 3 is the major integral protein on the red cell membrane that interacts with the membrane of band 3 is found in about 20% of American and European patients with but is more common in (Jarolim et al, 1996a; Eber et al, 1996; et al, 1994). The disease is as a dominant with anaemia and spherocytosis. patients also have a small of or et al, 1994; Jarolim et al, which are not in other forms of HS. A model of band 3 deficiency showed that band 3 is for stability of the membrane lipid bilayer but not for of the membrane skeleton et al, 1996). in the band 3 gene that result in mutations have been in patients with HS. Band 3 and have nonsense mutations that cause mRNA and band 3 deficiency (Eber et al, 1996; Jarolim et al, 1996a; et al, 1996; et al, 1996). Band 3 and have in the region that a frameshift band 3 Napoli I, and have (Miraglia del Giudice et al, Dhermy et al, 1997). Band 3 and result from and band 3 of a in the gene et al, 1997; Jarolim et al, 1994). mutations or short have also been found in HS patients. mutations are in the transmembrane domain and probably cause of band 3 into the membrane. In band 3 I, II and of residues at the of transmembrane probably with of band 3 into the of the (Eber et al, 1996; Jarolim et al, Dhermy et al, 1997). In band 3 Napoli and other amino for of band 3 within the lipid are et al, 1995; et al, 1997; Miraglia del Giudice et al, A in the transmembrane domain in band 3 of the peptide into the membrane et al, 1997), and a amino acid in the transmembrane domain is found in band 3 II (Eber et al, 1996). mutations in the cytoplasmic domain of band 3 can with its binding to other membrane skeleton in a functional In band 3 a of amino from the site this binding (Jarolim et al, An amino acid in the cytoplasmic domain in band 3 protein 4.2 binding. A both band 3 and band 3 has of protein 4.2 et al, 1997). with band 3 and have haemolytic anaemia with protein 4.2 deficiency (Jarolim et al, et al, 1993). of these alleles have mutations in the cytoplasmic domain, but are manifest in with band 3 and in the with band 3 The for this is Band 3 has a in the of band 3, causing HS et al, 1997). Several band 3 alleles the severity of the Band 3 and which both have mutations in the cytoplasmic domain, the HS of band 3 either is A promoter mutation in band 3 increases the clinical severity of HS in a with band 3 et al, 1996). band 3 protein functions as an anion and is also in the cells in the patients with HS and band 3 deficiency have been for a in patients with band 3 have but most HS patients with band 3 deficiency have of et al, 1997; Jarolim et al, 1998). In patients with band 3 there is increased but et al, 1997). A model of band 3 deficiency et al, and there are in that have a of band 3 that both the red cell and et al, 1996). Band 3 mutations have been found in patients with dominant but these patients have red cell abnormality and the mutations are different from with HS (Jarolim et al, et al, 1997; et al, 1998). deficiency of protein 4.2 is in patients with deficient ankyrin or band 3, to the underlying (Lux et al, 1990; Lanciotti et al, 1997). Protein 4.2 is also in a model with of the band 3 gene et al, 1996). some HS patients have an isolated deficiency of protein 4.2 in their red cell The disease is but and blood smears of patients may show or in to spherocytosis. In cases haemolysis is et al, protein 4.2 mutations underlying the disease have been Protein 4.2 is a common mutation in Japan et al, It from a mutation that haemolytic disease in or in with a allele as protein 4.2 or et al, 1994; et al, 1995; et al, 1995b). The two protein 4.2 mutations the are protein 4.2 and found in the form in and patients, et al, b). Hereditary elliptocytosis and hereditary are a of red cell membrane disorders with a of clinical from elliptocytosis to haemolytic anaemia with and red cell The genetic basis of HE/HPP was in the past patients with with and the inheritance was not Protein analysis has shown that the underlying most cases of HE/HPP is a of spectrin heterodimers to self-associate into which are the of the membrane skeleton In normal the of in the red cell membrane is In HE/HPP patients the can be as high as and correlates with clinical severity et al, 1993; et al, 1997). In to the patients have some degree of spectrin deficiency et al, 1993). It is known that patients are for an for two alleles, or of one allele and a spectrin The mutations causing HE/HPP involve components of the membrane skeleton responsible for interactions in the horizontal α spectrin, β spectrin and protein 4.1 (Fig 1). the structural integrity of spectrin are The of the spectrin peptide is into triple helical coiled-coil repeat (Fig The β and α spectrin side-to-side into a in a with a the of β spectrin and the of α spectrin et al, The β and α spectrin heterodimers self-associate into a by a head-to-head interaction involving the of β spectrin and the of α The β and α two and one to form a triple helical repeat which the spectrin site et al, 1990; et al, 1993). The of these repeat is for the integrity of the spectrin and most mutations found in HE/HPP to this . of the spectrin the triple helical coiled-coil repeats of the spectrin the head-to-head spectrin and the that the side-to-side interaction between α and β spectrin than mutations have been that cause or causing frameshift mutations and premature termination of β spectrin are found in and et al, 1994). site mutations in and and a nonsense mutation in spectrin result in truncated β spectrin in et al, 1996). mutations in β spectrin are present in and in which amino acid either the triple helical coiled-coil structure that forms the spectrin site or residues critical to the interaction et al, 1990; et al, 1993; et al, 1994; Gallagher et al, 1997; et al, 1996; et al, 1997; et al, 1998). these mutations the site of β spectrin (Fig 2). A more mutation cause mRNA or protein in spectrin deficiency and a of HS This is in the of in which patients have a clinical between HS and In and which are with mutations in β spectrin result in truncated that are both unstable and in (Jarolim et al, et al, 1997). The spectrin deficiency and may the mutations in α spectrin have been found in patients with of the mutations are amino acid the few repeat of α spectrin, to the site of (Fig 2). Several are and de et al, 1993; Gallagher et al, 1993; et al, 1993; et al, 1994; et al, 1994; et al, 1995). A few other mutations involve of short of the In spectrin a site mutation in a amino acid in the and in spectrin a in the α spectrin gene of residues in the repeat et al, Hassoun et al, 1994). These mutations the of the α spectrin peptide and with its to interact with β In the the mutation is from the spectrin site the is its In spectrin for a site mutation an of of the α spectrin which is from the site. of the mutation are and the has elliptocytosis for a et al, 1993). the of β spectrin, few mutations that result in truncated α spectrin have been in patients with a peptide the spectrin site and probably not be into the membrane. α spectrin is in to β spectrin et al, 1998). the allele is it interacts with other spectrin mutations and may for of the clinical in the HE/HPP α spectrin are in and the mutation of is by the allele in to a allele a α spectrin, however, the peptide with the structural will be into the membrane, the severity of the disease et al, As an a with a de novo mutation with of elliptocytosis also an allele in to the allele et al, 1993). In the with spectrin the haemolysis and several other affected elliptocytosis et al, Molecular analysis showed that the the allele from and an allele from which more clinical The of the can also be The allele in the same as an ameliorates the severity of the the peptide the is not into the membrane et al, 1994). a in which the red cell membrane contains a reduced of protein show elliptocytosis but haemolysis or membrane present with haemolytic anaemia with and erythrocytes. molecular defects have been in different In 4.1 and 4.1 a mutation in the of the protein et al, et al, 1995). A in 4.1 the in protein 4.1 deficiency in the et al, 1993). In protein 4.1 a of the gene et al, 1998). of a in the spectrin-binding domain in protein 4.1 its to spectrin et al, 1994). other membrane skeleton mutations that other than the of protein 4.1 have not been is a form of hereditary elliptocytosis found in and the of blood or some of which have one or two that the central et al, 1998). These red cells are not in any other with are but a few affected have varying degrees of haemolysis et al, 1993; et al, 1996). is probably et al, 1994; et al, 1995). In to other forms of is with increased and decreased of the red cell membrane. The molecular is a of amino residues at the of the cytoplasmic and transmembrane domains of band 3 (Jarolim et al, band 3 has decreased anion et al, and an increased to form in the membrane et al, 1995). The of with et al, and there is a of prevalence in patients with disease severity et al, that the recent a new form of elliptocytosis that is different from other known types et al, 1998). in an have a of band in the affected have to of the gene in The two affected also have there is of or erythrocyte membrane These findings the of a elliptocytosis on the identification of which will our understanding of that the of erythrocytes. The recent of the molecular defects in many patients with hereditary spherocytosis and hereditary the molecular of these and are caused by defects in the horizontal interactions that the membrane skeleton the critical spectrin gene defects cause red cells to as by a and are The of two genes or one α spectrin and a in the allele in cells that into in the with haemolysis and A few of the α spectrin defects are an or some other in is caused by These involve the proteins that the membrane skeleton to the lipid α and β spectrin, ankyrin, band 3 and protein 4.2. loss of the band 3 its lipid et al, of the membrane surface leading to and will to on each of its the membrane skeleton defects red cells are and in the and patients with or defects can have different clinical also to be to for and new for which can patients from and red cell and from & and et al, 1996). This work was in by from the of