Genomic DNA from five kindreds and two individuals with hereditary elliptocytosis [HE(4.1+)] and a partial deficiency of protein 4.1 [HE(4.1+)] was extracted and probed with a cDNA for protein 4.1. When using a fragment of the cDNA that encompassed the coding region of the gene, two restriction fragment length polymorphisms segregating with protein 4.1 deficiency were found in one kindred when using the enzymes BgIII and PvuII but were not seen in the other HE(4.1+) subjects or in 20 random control individuals. DNA digested with three other enzymes (HindIII, EcoRI, TaqI) produced restriction patterns similar to controls. The unique BgIII and PvuII polymorphisms probably reflect a rearrangement of the coding region of the protein 4.1 gene as the underlying cause of the partial protein 4.1 deficiency in this family. A less likely possibility is that these polymorphisms represent coincidental single base changes unrelated to the primary gene defect.

Protein 4.1, an important component of the red cell membrane skeleton, was quantitated relative to protein 3 after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of membranes isolated from red cells of members of 14 kindreds with hereditary elliptocytosis (HE) who reside in South Africa. A partial deficiency of protein 4.1 (mean 30% reduction) was inherited in autosomal dominant fashion in five white kindreds giving a frequency of 0.36 of HE families studied. Immunoblots of membrane proteins separated by SDS-PAGE and probed with a monoclonal antibody to protein 4.1 did not reveal any proteolytic fragments in the 4.1-deficient subjects that could account for the reduction of this protein. These studies draw attention to the relatively high frequency of this condition as a cause of HE in white subjects in this country.

A kindred is described in which two brothers with a poikilocytic variant of hereditary elliptocytosis (HE) were found to have a defect of spectrin dimer association and a decreased spectrin-band 3 ratio. Two-dimensional gel electrophoresis of limited tryptic digests of their spectrin revealed decreased amounts of the alpha I domain when compared with control digests and the appearance of two major peptides with mol wts of 43,000 and 42,000 and isoelectric points (5.75 to 5.85) more basic than the alpha I domain. Tryptic digests of spectrin from the asymptomatic mother of the two brothers were normal. Immunoblots of the two-dimensional gels using an antiserum to the alpha I domain revealed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain, along with a series of lower mol wt peptides, some of which were below the detection limits of Coomassie blue-stained gels. Limit chymotryptic maps of 125I-labeled tryptic peptides confirmed that the 43,000- and 42,000-dalton peptides were derived from the alpha I domain. This kindred represents a new structural variant of spectrin in HE in that the major abnormal tryptic peptides derived from the alpha I domain have lower mol wts and more basic isoelectric points than hitherto described.

Following restricted tryptic digestion at 4 degrees C, a structural polymorphism affecting the alpha-chain of human spectrin, the major erythrocyte membrane skeleton protein, has recently been described in American blacks (Knowles, W.J., Bologna, M.L., Chasis, J.A., Marchesi, S.L. and Marchesi, V.T. (1984) J. Clin. Invest 73, 973-979). Four variants affecting the alpha-II domain or its tryptic products have been characterized, depending on changes in molecular weight and/or isoelectric point. One variant of the alpha-II domain (Type 2) shows an increase in apparent molecular weight and basic shift in pI. It contains a limit chymotryptic peptide showing a change in chromatographic mobility on two-dimensional electrophoresis which is thought to reflect a sequence alteration associated with the increase in apparent molecular weight. We find that this altered limit chymotryptic peptide is not unique to the Type 2 variant, but is also present in a variant (Type 4) showing only the same basic shift in pI as the Type 2 variant. It is not found in a variant (Type 3) showing only an increase in apparent molecular weight. The most likely explanation for these findings is that the altered limit chymotryptic peptide common to both the Type 2 and Type 4 variants is responsible for the change in isoelectric point which is common to both these variants. An as yet unidentified change elsewhere in the polypeptide chain must be responsible for the observed alteration in molecular weight of the Types 2 and 3 variants.

The lipid bilayer of the adult red cell is supported on its inner surface by a complex arrangement of proteins known as the membrane skeleton. This filamentous network, a major component of which is a multifunctional protein called spectrin, has an essential role in determining the shape, structural integrity, and deformability of the red cell. A significant achievement of modern biochemistry and hematology has been the elucidation of the organization of the components of the membrane skeleton and their relationship to other membrane proteins and lipids. This article reviews current concepts of membrane skeleton structure and function and emphasizes recent advances which have been made in characterizing and classifying molecular defects of the skeleton which manifest clinically with changes in the shape and stability of the red cell. The pathobiology of hereditary skeletal defects associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) are comprehensively discussed. Secondary defects of the membrane skeleton occurring in glucose-6-phosphate dehydrogenase deficiency and sickle cell anemia are also briefly considered.

The proportion of spectrin tetramers and dimers in 4 degrees C low ionic strength extracts of red cell membranes of 9 subjects with 4 different variants of hereditary elliptocytosis (HE) and 2 subjects with hereditary spherocytosis (HS) was determined by nondenaturing gel electrophoresis. Such extracts reflect the native oligomeric state of spectrin in the red cell membrane. In two hemolytic HE variants (an unclassified adult with increased thermal sensitivity of red cells and an infant also showing increased thermal sensitivity of red cells), the proportion of dimers was increased, whereas the remaining subjects had values within the control range. Conversion of spectrin tetramers to dimers under isotonic conditions at 37 degrees C, or spectrin dimers to tetramers at 30 degrees C, resulted in a high proportion of dimers in the above two HE variants, as well as in a third variant with probable mild HE and sporadic hemolysis. The mother of the infant with elliptocytosis and increased thermal sensitivity of red cells, although hematologically normal, had an increased proportion of dimers in 4 degrees C low ionic strength extracts of her red cell membranes. These findings reflect an underlying primary or secondary abnormality of spectrin in these subjects that affects the association state of spectrin in the red cell membrane. Their exact relationship to the pathogenesis of the elliptical shape of the red cell, or to the presence of hemolysis, is at present unclear.

G6PD-deficient red cells (nonhemolytic variants) incubated for 24 hr in medium containing glucose maintain their adenosine triphosphate (ATP) levels but show a pronounced fall in reduced glutathione (GSH) levels. Membranes of such ATP-replete, GSH-depleted red cells contain a high molecular weight protein aggregate that is disulphide bonded. This aggregate is similar in composition to that found in control red cells that are incubated in the absence of substrate (i.e., ATP- and GSH-depleted) in that considerable amounts of spectrin are present with a high spectrin:band 3 ratio. These cells also show a marked decrease in spectrin “extractability” and do not show any overt shape change on phase-contrast microscopy. Addition of acetylphenylhydrazine to the medium results in a high molecular weight aggregate consisting primarily of globin, although such cells are also ATP-replete and GSH-depleted. Our findings indicate that spectrin rearrangement can occur in ATP-replete red cells, but that such cells can still maintain their discoid shape. Addition of autologous plasma or albumin to substrate-containing medium in which G6PD-deficient cells are incubated has a marked protective effect on membrane protein aggregate formation. This effect is mediated across a dialysis membrane. Although the exact mechanism is unclear, the protective effect of albumin does not appear to be mediated by scavenging ambient oxidants or binding trace amounts of metals. These in vitro studies support recent concepts of mechanisms of hemolysis in hemolytic G6PD variants in which normal red cell ATP levels and spectrin-containing membrane aggregates have been detected.

British Journal of HaematologyVolume 10, Issue 1 p. 50-58 Iron Metabolism in Scurvy with Special Reference to Erythropoiesis* T. H. Bothwell, Corresponding Author T. H. Bothwell Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, Johannesburg†Hospital Street, Johannesburg, S. Africa.Search for more papers by this authorB. A. Bradlow, B. A. Bradlow Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorP. Jacobs, P. Jacobs Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorK. Keeley, K. Keeley Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorS. Kramer, S. Kramer Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorH. Seftel, H. Seftel Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorS. Zail, S. Zail Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this author T. H. Bothwell, Corresponding Author T. H. Bothwell Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, Johannesburg†Hospital Street, Johannesburg, S. Africa.Search for more papers by this authorB. A. Bradlow, B. A. Bradlow Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorP. Jacobs, P. Jacobs Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorK. Keeley, K. Keeley Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorS. Kramer, S. Kramer Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorH. Seftel, H. Seftel Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this authorS. Zail, S. Zail Departments of Medicine and Chemical Pathology, University of Witwatersrand Medical School, and Baragwanath Hospital, JohannesburgSearch for more papers by this author First published: January 1964 https://doi.org/10.1111/j.1365-2141.1964.tb00677.xCitations: 35 * This work was supported in part by a grant (AM–04912–02) from the National Institutes of Health, U.S.A., and in part by a grant from the Council for Scientific and Industrial Research, South Africa. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume10, Issue1January 1964Pages 50-58 RelatedInformation