In overhydrated hereditary stomatocytosis (OHSt), Coomassie-and silver-stained polyacrylamide gels show an apparently complete deficit of the 32-kDa membrane protein, stomatin. We have used an antistomatin antibody to examine peripheral blood films, bone marrow, splenic tissue, and hepatic tissue from these patients by immunocytochemistry. This technique revealed that, in fact, some red cells did show positive stomatin immunoreactivity; and consistent with this result, Western blot analysis of the red cell membranes confirmed that about one twentieth to one fiftieth of the normal amount of stomatin was in fact present. Flow cytometry, combining immunoreactive quantitation of stomatin expression with thiazole orange staining for reticulocytes, showed that in OHSt, it was the young cells that had more stomatin. Magnetic-activated cell separation studies, using beads to which an anti-transferrin receptor antibody was conjugated, confirmed that in OHSt there was a correspondence between expression of stomatin and the transferrin receptor. Immunocytochemistry and Western blotting revealed that in OHSt patients, the protein was present in spleen, liver, neutrophils, platelets, monocytes, and about 50% of the peripheral lymphocytes, with the same distribution as in healthy controls. Neither Southern blots, nor direct sequencing of multiple subclones of the cDNA, nor sequencing of amplicons from genomic DNA revealed any significant abnormality in stomatin gene sequence in these patients. The deficiency of stomatin from red cells appears to be due to a loss of stomatin from these red cells on maturation in the bone marrow and in the circulation. IntroductionThe name "stomatocytosis" was coined to describe the morphology in a dominantly inherited hemolytic anemia distinguished from hereditary spherocytosis by the cell shape. 1 This original condition is now known as overhydrated hereditary stomatocytosis (OHSt). Aside from the morphology, the anemia shows 2 other features: a catastrophic "leak" across the plasma membrane to the univalent cations Na ϩ and K ϩ (Zarkowsky et al 2 ), and the deficiency of the 32-kDa integral membrane protein, stomatin, or erythrocyte membrane protein 7.2b. [3][4][5] The condition represents an unusual genetic situation, in which a protein is apparently missing in a dominant, and therefore presumably heterozygous, condition. The gene coding for the missing protein has been cloned and sequenced. 5,6 Preliminary reports have indicated that no mutation has been found in American OHSt families, in the protein-coding region at least. 7,8 The pedigrees of OHSt are too small for effective genome-wide searches, but other variants of this leaky-cell class of red cell disease have been mapped. An American pedigree with nonstomatin-deficient dehydrated hereditary stomatocytosis (HSt) did not map to the stomatin locus on chromosome 9, 9,10 and European pedigrees of this non-stomatin-deficient dehydrated condition map to chromosome 16. 11 Other HSt pedigrees with different variants, none of which showed s...

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In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.

Summary. The hereditary stomatocytoses are a group of dominant haemolytic anaemias that show two main features: invaginated, 'stomatocytic' morphology; and a membrane leak to the univalent cations Na and K. A patient with the most severe variant of these conditions was reported to show a defect in an in vitro process of ATPdependent endocytic vesiculation (ADEV), which is found in normal red cells. We have examined this endocytosis process in 11 leaky red cell pedigrees available to us in the UK. ADEV in broken membranes was absent only in the two most severely affected, 'overhydrated' pedigrees studied, both of which showed a deficiency in the membrane raft protein, stomatin. The process was present, although typically diminished by about 10-20% compared with normal red cells, in all others. The cross-linker dimethyl adipimate (DMA), which could correct the cation leak in some of these patients, also corrected the ADEV defect in the same patients. In those patients in whom DMA had no effect on the ion leak, ADEV was not absent. In normal cells, this process of vesiculation was inhibited by inhibitors of membrane 'raft' function, by an antistomatin antibody and by vanadate and N-ethyl maleimide, but not by inhibitors of a number of kinases. These data highlight the heterogeneity of these conditions. A mechanism is discussed by which a defect in raft-based endocytosis could lead to the exaggerated surface exposure of an ion channel, which could then function constitutively, i.e. 'leak'.