The possibility that the primary lesion of HE and HPP erythrocytes resides in the proteins of the RBC membrane skeleton was first raised by the findings of thermal instability of HPP spectrin, retention of the elliptical shape in HE membrane skeletons, disintegration of mem brane skeletons after exposure to shear stress, defective self-association of spectrin dimers to tetramers, altered susceptibility of spectrin to tryptic digestion, and a deficiency of the membrane skeleton proteins spectrin and protein 4.1R. Gene cloning and determination of the primary structure of these proteins was soon followed by reports of mutations in the genes encoding erythrocyte membrane proteins.
Spectrin Mutations
The most common defects in HE, found in approximately two-thirds to three-quarters of all patients, are mutations of α- or β-spectrin. Both α- and β-spectrin are elongated flexible molecules consisting of triple-helical repeats connected by nonhelical segments. These poly peptides are associated side to side in an antiparallel position, forming a flexible, rod-like αβ heterodimer in which the NH2-terminal of α-spectrin and the COOH-terminal of β-spectrin form the head region of the heterodimer. Spectrin heterodimers associate head to head to form spectrin tetramers, the major structural subunits of the membrane skeleton. Spectrin tetramers in turn are interconnected into a highly ordered two-dimensional lattice through binding, at their distal ends, to actin oligomers with the aid of protein 4.1R.
The contact site between the α- and β-spectrin chains of the opposed heterodimers is a combined “atypical” triple-helical repetitive segment in which the first two helices are contributed by the COOH terminal of β-spectrin, whereas helix 3 is the first helical segment of α-spectrin. Spectrin dimer-tetramer interconversion is governed by a simple thermodynamic equilibrium that under physiologic conditions strongly favors spectrin tetramers. Most α-spectrin defects are at or near the NH2-terminal of α-spectrin, which is involved in the heterodimer contact (the αI domain defined by limited tryptic peptide mapping; see the discussion under Laboratory Manifestations), and impair the self-association of spectrin into tetramers. Most α-spectrin mutations are point mutations. These mutations create abnormal proteolytic cleavage sites that typically reside in the third helix of a repetitive segment and give rise to abnormal tryptic peptides on two dimensional tryptic peptide maps of spectrin.
Elliptocytogenic β-spectrin mutations are COOH-terminal point mutations or truncations that disrupt the formation of the combined β triple-helical repetitive segment and consequently the self-association of spectrin heterodimers to tetramers. All of these mutations open a proteolytic cleavage site residing in the third helix of the combined repetitive segment, giving rise to a 74-kDa αI peptide.
Although most spectrin mutations reside in the vicinity of the αβ-spectrin self-association site, a few mutations remote from the self-association site have been described. These mutations are asymptomatic in the simple heterozygous state but cause hemolytic anemia, which can be severe, in homozygous patients. Unlike mutations located in the self-association contact site, which are predicted to disrupt the conformation of the local protein structure, mutations outside this region are predicted to perturb long-range protein-protein interactions, disrupting the positively coupled, cooperative interactions of αβ spectrin self-association, spectrin-ankyrin inter actions, and ankyrin–band 3 interactions. One HE-associated mutation in a linker region remote from the self-association contact site disrupted the stability propagated from one spectrin repeat to the next.
Protein 4.1R Mutations
Another group of elliptocytogenic mutations, although much less common than spectrin mutations, are quantitative or qualitative defects of protein 4.1R. Protein 4.1R is a multifunctional protein that contains several important sites of protein interactions, including the spectrin binding domain, where 4.1R binds to the distal end of the spectrin αβ heterodimer, markedly increasing the binding of spectrin to oligomeric actin, and the basic NH2-terminal domain, where 4.1R interacts with GPC, phosphatidylinositol, and phosphatidylserine, facilitating the attachment of the distal end of spectrin to the membrane.
Studies of 4.1R mRNA from normal RBCs revealed 4.1R isoforms resulting from complex tissue- and developmental stage-specific pat terns of alternate mRNA splicing. Alternate translation initiation sites are present in the protein 4.1R mRNA. When an upstream initia tor methionine is used, isoforms greater than 80kDa are synthesized. During erythropoiesis, this upstream initiator methionine is spliced out and a downstream initiator methionine is used, leading to the production of the 80-kDa mature erythroid protein 4.1R isoform. On SDS-PAGE, protein 4.1R is resolved into two bands of different sizes: 4.1a and 4.1b. The larger band, 4.1a, is typically found in normal RBCs, whereas the shorter one, 4.1b, represents the major isoform of reticulocytes. The 4.1b isoform is converted into the 4.1a isoform by deamidation of Asn 502.
A partial deficiency of protein 4.1R is associated with mild, dominantly inherited HE, whereas a complete deficiency (a homozygous state) leads to a severe hemolytic disease. Homozygous protein 4.1R(−) erythrocytes fragment more rapidly than normal at moderate shear stresses, an indication of their intrinsic instability. Membrane mechanic stability can be restored by reconstituting the deficient RBCs with protein 4.1R or the protein 4.1R/spectrin/actin-binding site. Homozygous protein 4.1R(−) erythrocytes also lack p55 and have only 30% of the normal content of GPC. These homozygous protein 4.1R(−) erythrocytes, as well as GPC-deficient Leach erythrocytes, demonstrate decreased invasion and growth of Plasmodium falciparum in vitro.
Mutations associated with protein 4.1R deficiency have included deletions that include the exon encoding the erythroid transcription start size and mutations of the transcription initiation codon. Qualitative defects of protein 4.1R protein include deletions and duplications of the exons encoding the spectrin-binding domain, leading either to truncated or elongated forms of protein 4.1R. Electron microscopic studies of homozygous protein 4.1R(−) erythrocyte mem branes revealed a markedly disrupted skeletal network with disruption of the intramembrane particles, suggesting that protein 4.1R plays an important role in maintenance not only of the skeletal network but also of the integral proteins of the membrane structure.
Glycophorin C Deficiency
GPC has been found absent because of a variety of molecular defects. In contrast to other forms of HE, which are dominantly inherited, heterozygous carriers are asymptomatic, with normal RBC morphology, and homozygous patients have no anemia and only mild elliptocytosis apparent on the peripheral blood film.
GPC deficiency with elliptocytosis, the so-called Leach phenotype, caused by reduced expression of GPC, should be distinguished from the immunochemically defined phenotypes Gerbich and Yus, in which abnormal glycoproteins are formed that can functionally substitute for normal GPC and preserve the normal RBC shape. The Leach phenotype is usually caused by a large deletion of genomic DNA (~7 kb) that removes exons 3 and 4 from the GPC/glycophorin D locus. In one patient, the Leach phenotype was caused by a frame shift mutation.
GPC-deficient patients are also partially deficient in protein 4.1R and lack p55, presumably because these proteins form a complex and recruit or stabilize each other on the membrane. It has been speculated that the protein 4.1R deficiency in Leach erythrocytes is the cause of the elliptocytic shape. In contrast, patients deficient in glycophorin A, the major transmembrane glycoprotein, are asymptomatic.
