Abnormalities of the red cell membrane

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CHAPTER 7 Abnormalities of the red cell membrane

A number of hereditary hemolytic anemias result from mutations affecting the quality and/or amount of proteins that belong to the red cell membrane, its skeleton, or the attachment systems (nexuses) of the latter to the former. Most proteins participate in complexes (Fig. 7.1). They play a role in erythrocyte resilience and elastic deformability, either mechanically, through the skeleton and its attaching systems, or osmotically, through a variety of transporters and pumps. Major proteins and their genes are listed in Table 7.1. The ever increasing number of mutations, too numerous to detail here, have given insight into the function of such protein domains and the regulatory regions of some genes. A selection of abnormally-shaped red cells is shown in Fig. 7.2.

Fig. 7.1 Major proteins of the red cell membrane and their organization in complexes. The major proteins, usually belonging to complexes, are represented. Not all proteins mentioned are shown. Box A: the spectrin self-association site. Spectrin α₂β₂ tetramers form a network lining the inner surface of the lipid bilayer. The α- and β-chains are antiparallel and contain 22 and 17 repeats, respectively. Two dimers associate side-by-side, a process set off at the nucleation sites on both chains, near the C– and N-terminal regions of the α- and β-chains, respectively. Dimers associate head-to-head, α-chain N-terminal region vs. β-chain C-terminal region, at the self-association site, in order to generate tetramers, or higher order oligomers. Spectrin, through its α4 repeat (away from Box A), interacts with the Lu-BCAM protein. Box B: the 4.1R-based multiprotein complex: (i) the junctional complex. Several converging spectrin tetramers interact with oligomeric β-actin, whose length is limited by tropomodulin. 4.1R strengthens this interaction through its 10 kDa domain, which binds to a site located in the spectrin β-chain N-terminal region. Many additional proteins participate in the junctional complex: dematin (protein 4.9), tropomyosin, α- and β-adducin, and several others. Box C: the 4.1R-based multiprotein complex: (ii) the 4.1R-glycophorin C/D-p55 complex. 4.1R interacts through its 30 kDa domain with transmembrane glycophorin C/D and p55 in a triangular fashion. Box D: the band 3-based multiprotein complex: (i) the band 3 complex, stricto sensu. Band 3 appears as a tetramer. The bulky part of each band 3 monomer represents 12 transmembrane segments of band 3. The stalky part accounts for its cytoplasmic domain which serves as an anchor to ankyrin-1, protein 4.2 and many cytoplasmic proteins. Ankyrin-1, in turn, binds to spectrin β-chain (C-terminal region). Recently, band 3 has also been demonstrated to be present in the 4.1R multiprotein complex, making the interactions much more complicated (not shown). Box E: the band 3-based multiprotein complex: (ii) the Rh complex. It includes the Rh polypeptides (RhD/RhCE) and the RhAG protein (Rh-associated glycoprotein), being arranged as a trimer; the latter is associated with CD47, the Landsteiner–Wiener glycoprotein (LW, also called ICAM-4) and glycophorin B.

Table 7.1 Major membrane proteins, their genes and related diseases

Fig. 7.2 (A–F) Selected examples of red cell morphological abnormalities. (A) Moderate dominantly inherited spherocytosis (mutation unknown). (B) Mild, recessively inherited spherocytosis due to the total absence of protein 4.2. (C) Severe, recessively inherited hereditary spherocytosis, with a strong touch of poikilocytosis (mutations in the SPTA1 gene). (D) Asymptomatic, dominantly inherited elliptocytosis due to the partial absence of 4.1R. (E) Dehydrated hereditary stomatocytosis (gene unknown). (F) Overhydrated hereditary stomatocytosis (mutation in the RHAG gene). May–Grünwald–Giemsa. × 1000.

(Courtesy of Dr Thérèse Cynober.)

It should be pointed out that although the genes involved are expressed in a wide range of cell types as isoforms (spliceoforms in particular), genetic disorders are usually confined to the erythroid line. Naturally affected animals and animals with experimentally invalidated genes are helpful, although they do not necessarily mirror the human diseases.

Hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common genetic disorder of the red cell membrane in Western countries. Its incidence has been estimated as 1 in 2000 live births and there is a wide spectrum of clinical severity. In typical cases the hemolytic anemia is moderate, with an increased reticulocyte count a reticulocytosis, intermittent jaundice, gallstones and splenomegaly. Severe cases are rare and may cause death in utero or shortly after birth. In contrast, patients with mild HS may be over 60 years of age when diagnosed. Parvovirus B19 infection commonly occurs. Blood films show a variable percentage of spherocytes. The diagnosis relies on an increased percentage of hyperdense cells and a reduction in osmotic resistance, and on a number of tests, including polyacrylamide gel electrophoresis of the red cell membrane proteins in the presence of sodium dodecylsulfate (SDS-PAGE). The main treatment is splenectomy, though the need for this should be carefully weighed owing to its complications, namely severe infections and a statistically significant increase in thromboembolic accidents.¹ Transfusions may be necessary.

Most cases of HS result from reduced or absent proteins. Consequently, the lining of the inner surface of the lipid bilayer by the skeletal meshwork is less dense. Microvesicles bud out of naked bilayer patches. The surface area shrinks and the normal discoid cells gradually turn into spherocytes. The six genes most commonly involved in HS are discussed below.

ANK1 gene mutations

Ankyrin-1 is encoded by ANK1.² It connects the skeleton to band 3, i.e. the anion exchanger-1. Approximately 60% of HS are due to ANK1 gene mutations and have reduced ankyrin-1. HS due to ANK1 mutations is relatively severe and has a dominant inheritance pattern, although de novo mutations may occur. Homozygosity is bound to be lethal. (One case has been recently described, however.) This may not be evident due to the elevated reticulocyte count, as young cells have a higher ankyrin-1 content. Spectrin α- and β-chains, and protein 4.2, interacting with ankyrin-1, are secondarily decreased.

SLC4A1 gene mutations

Band 3, encoded by SLC4A1, is the pillar of the band 3 complex stricto sensu, which is itself attached to the Rh complex. Both complexes are linked through protein 4.2-CD47 ³ and Rh/RhAG-ankyrin-1 contacts.⁴ In the heterozygous state, mutations in SLC4A1 produce a mild, dominantly inherited HS (approximately 20% of HS cases). Band 3 is uniformly reduced, along with a proportional decrease in protein 4.2. Scores of mutations have been reported since the first report.⁵ More severe cases are seen in compound heterozygotes. Two homozygous cases, leading to missing or strongly reduced band 3, have been reported. They were associated with a dramatic picture. Spherocytes were replaced in part by poikilocytes (see below).^6,⁷ The absence of band 3 is likely to be lethal unless intensive care is provided prior to and following birth. An early subtotal splenectomy, to be completed later, is indicated. These cases are accompanied by distal renal tubular acidosis, due to the fact that α-intercalated cells of the distal tubule basolateral membrane contain an isoform of band 3, lacking the 65 first amino acids of the erythroid isoform. The prognosis is obscured by nephrocalcinosis. A third case of homozygosity was described free of renal disorders because the mutation lay in the missing region of the renal isoform of band 3.

SPTB gene mutations

SPTB encodes spectrin β-chain. Mutations generate a dominantly inherited, relatively severe form of HS (20% of HS in Europe).⁸ There is an isolated reduction in spectrin (α- and β-chains) on SDS-PAGE. De novo mutations may occur. Homozygous cases have never been reported.

EPB42 gene mutations

Mutations in EPB42, encoding protein 4.2, engender a relatively rare recessively inherited form of HS which is not absolutely typical. Spherocytes are bulky. A mutation has shown some frequency in Japan (Ala142Thr).⁹

SPTA1 gene mutations

SPTA1 encodes spectrin α-chain. Mutations in SPTA1 are rare and show a recessive inheritance pattern. Homozygosity, or compound heterozygosity (involving peremptory mutations: stop codon or frameshift), has never been reported. In contrast, compound heterozygosity for a peremptory HS mutation and a rather common weak allele of the SPTA1 gene, allele α^LEPRA, has been observed, causing a severe picture with poikilocytosis.¹⁰

RHAG gene mutations

RHAG encodes the Rh-associated glycoprotein (RhAG). The exceptional absence of RhAG (Rh-deficiency of the regulator type) results in a severe hemolytic anemia which has been classified as HS, although this issue has never been assessed thoroughly.

Hereditary elliptocytosis and poikilocytosis

Hereditary elliptocytosis (HE) has a much lower incidence in Caucasians than HS but occurs with the same frequency as HS in black Africans. The clinical phenotype is usually mild with peripheral blood elliptocytes but it can be moderately severe. In severe forms that achieve hereditary poikilocyotosis (HP), large red cell fragments are torn off, appearing as schistocytes and leaving erythrocytes with marked poikilocytosis.

SPTA1 gene mutations

SPTA1 mutations account for approximately 60% of HE and HP. Their mode of inheritance is dominant. Mutations are located from the N-terminal region of the spectrin α-chain down to repeat α9.¹¹ As a consequence, the self-association process is impaired, loosening the meshwork at a critical junction. Homozygosity or compound heterozygosity has a moderate to severe presentation depending on the mutation(s). Severe forms display an HP phenotype. The same situation happens when an HE mutation lies in trans to a frequent, worldwide, low-expression allele of the SPTA1 gene, allele α^LELY.¹² Splenectomy should only be considered in the most severe cases.

SPTB gene mutations

SPTB mutations are rarer than SPTA1 mutations and are associated with HE or HP. They are sporadic and dominantly transmitted. Mutations are situated in the C-terminal region (repeat β17) of the spectrin β-chain, in the self-association site, and with the same consequences as the facing mutations in the α-chain.¹¹ Homozygosity may be life-threatening.

EPB41 gene mutations

The EPB41 gene encodes protein 4.1R. Mutations in EPB41 account for 20–30% of HE in Caucasians. In the heterozygous state, 20–30% of 4.1R is missing. The 4.1R (−) trait, which is dominantly transmitted, is symptomless. In the homozygous state,¹³ in which 4.1R is absent, HP is severe in the newborn but tends to improve later on. Early subtotal splenectomy may accelerate the improvement.

Genetic disorders of the monovalent cation leak across the membrane

Disorders of monovalent cation leak across the membrane, or cation leak, encompass conditions that remain poorly understood. Most include hemolytic anemia, jaundice and splenomegaly, erythrocyte shape abnormalities and macrocytosis. They have a major tendency to iron overload, an alteration of intra-erythrocytic cation concentrations, and an increase in the cation leak. The leak is defined as the fluxes that remain when the Na⁺, K⁺ ATPase and the Na⁺, K⁺, 2Cl⁻ co-transporter are inhibited by ouabain and bumetanide, respectively. The leak assumes various curves as a function of temperature. In a subset of cases, the leak diminishes with a fall in temperature from 37°C to 20°C, and then resumes, sometimes dramatically, at lower temperatures, warranting the prefix ‘cryo-’. The inheritance pattern is dominant. Splenectomy is contraindicated in most of these conditions due to the risk of thromboembolic accidents, which may be lethal.¹⁴

A pleiotropic syndrome revolving around dehydrated hereditary stomatocytosis

Dehydrated hereditary stomatocytosis (DHSt) may occur alone, as first reported by Oski et al.¹⁵ The presentation is mild and sometimes revealed only at a late stage by its major complication, an iron overload (hemochromatosis). Anemia is well compensated, and the reticulocyte count is elevated. Stomatocytes are scarce and incompletely formed. There is a borderline macrocytosis. The osmotic resistance is increased. De novo mutations may occur. In the homozygous state it is lethal. The curve : cation leak as a function of temperature is monophasic and has a ‘shallow’ slope. One responsible gene has been mapped to 16q23-ter in a large Irish kindred.¹⁶ The region of interest was recently narrowed down to 16q24.1-ter in an extended French kindred.¹⁷ However, there must be another or other gene(s) involved. DHSt is associated with pseudohyperkalemia and/or perinatal fluid effusions, resulting in a pleiotropic syndrome.¹⁸

Pseudohyperkalemia designates an increase in potassium when collected blood is left at room temperature for a few hours. DHSt-related pseudohyperkalemia is similar to familial pseudohyperkalemia (FP), a dominantly inherited trait.¹⁹ Although extremely rare, it has been mapped to 16q23-ter²⁰ and to 2q35-36²¹ in distinct (Scottish and Flemish) extended families. The first location coincides with that of some cases of DHSt, strengthening the idea that FP is a truncated form of the pleiotropic syndrome.

Perinatal fluid effusion is characterized by mild (sonography discovery) to dramatic edema (hydrops fetalis). When severe, the edema must be treated in order to ease mechanical constraints on the fetus. These effusions, which are sometimes chylous, recede prior to birth or in the following months, never to reappear. Whether DHSt-related fluid effusions can occur alone has not been systematically investigated.

Overhydrated hereditary stomatocytosis

Overhydrated hereditary stomatocytosis (OHSt) is an exceedingly rare form of stomatocytosis. The first case of stomatocytosis ever described was an OHSt.²² The presentation is quite pronounced with anemia (sometimes requiring transfusions), marked macrocytosis, a strong tendency to iron overload and a reduced osmotic resistance. Stomatocytes are numerous and fully fledged. De novo mutations are frequent. A salient feature is the sharp decrease in, or the absence of, stomatin (not shown in Fig. 7.1). The responsible gene is RHAG.²³

Hereditary cryohydrocytosis

Hereditary cryohydrocytosis with normal stomatin (CHC1) is also an exceedingly rare disorder, first described by Miller et al.²⁴ CHC 1 presents like a stomatocytosis (though of the dehydrated type, with elevated mean corpuscular hemoglobin concentration (MCHC)). Pseudohyperkalemia may be present. The salient feature is the resumption of the leak at low temperature. Mutations have been found in the SLC4A1 gene.²⁵ Rare, atypical forms of HS (on the basis of the curve : leak as a function of temperature) displayed mutations in the involved region of band 3 (but with no reduction, as seen above); they were eventually related to CHC1.²⁵

Hereditary cryohydrocytosis with low or missing stomatin (CHC2) presents as OHSt. Only two cases have been reported worldwide.²⁶ Various neurological signs were found to be associated with the condition. There was a soaring resumption of the leak at low temperatures. No mutations were found in the RHAG gene.