Overhydrated Hereditary Stomatocytosis Research Articles

Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells.

Pannexin 1 (PANX1) was proposed to drive ATP release from red blood cells (RBCs) in response to stress conditions. Stomatin, a membrane protein regulating mechanosensitive channels, has been proposed to modulate PANX1 activity in non-erythroid cells. To determine whether stomatin modulates PANX1 activity in an erythroid context, we have (i) assessed the in situ stomatin-PANX1 interaction in RBCs, (ii) measured PANX1-stimulated activity in RBCs expressing stomatin or from OverHydrated Hereditary Stomatocytosis (OHSt) patients lacking stomatin, and in erythroid K562 cells invalidated for stomatin. Proximity Ligation Assay coupled with flow imaging shows 27.09% and 6.13% positive events in control and OHSt RBCs, respectively. The uptake of dyes 5(6)-Carboxyfluorescein (CF) and TO-PRO-3 was used to evaluate PANX1 activity. RBC permeability for CF is 34% and 11.8% in control and OHSt RBCs, respectively. PANX1 permeability for TO-PRO-3 is 35.72% and 18.42% in K562 stom+ and stom− clones, respectively. These results suggest an interaction between PANX1 and stomatin in human RBCs and show a significant defect in PANX1 activity in the absence of stomatin. Based on these results, we propose that stomatin plays a major role in opening the PANX1 pore by being involved in a caspase-independent lifting of autoinhibition.

Phenotype-Genotype Spectrum of Stomatocytic Disorders Encountered in India Using Next Generation Sequencing

IntroductionStomatocytes in peripheral blood are pathognomonic findings in multiple conditions along with hemolysis and reticulocytosis, often suggestive of erythrocyte membrane transport defects. These uncommon disorders are usually difficult to diagnose due to a wide range of overlapping phenotypes and a perception of stomatocytes being artefacts. Genes involved in these disorders are multiple (RHAG,SLC4A1, ABCG5, ABCG8, PIEZO1,KCNN4, ABCB6, SLC2A1 etc) rendering Sanger sequencing costly and labor intensive. Phenotypes vary from transfusion-dependent anemia to compensated hemolysis.MethodsSeventeen patients were encountered in 12 families and enrolled in this study. Majority of the cases showed the presence of significant numbers of red blood cells showing stomatocytes with or without thrombocytopenia. Various hematological, biochemical and molecular tests were used to exclude thalassemia syndromes and hemoglobinopathies, glucose-6-phosphate dehydrogenase (G6PD) deficiency, autoimmune hemolytic anemia, hereditary spherocytosis (HS) and pyruvate kinase deficiency. Genomic DNA was extracted by the QIAamp DNA Blood Midi Kit and quantified on NanoDrop 2000 spectrophotometer and QubitFluorometer. DNA libraries were prepared using Illumina's custom panels (TruSight One Sequencing Panel and TruSeq Custom Amplicon v1.5) and sequenced on a MiSeq Sequencing System. MiSeq Reporter and VariantStudio were used for analysis, classification, and reporting of variants. Variants which were predicted pathogenic by in silico analysis using PolyPhen-2, SIFT, PROVEAN (http://provean.jcvi.org/), Mutpred (http://mutpred.mutdb.org/) and Human Splicing Finder as indicated, were subjected to Sanger sequencing in the patient and family members (where available).ResultsOf these 17 patients, 10 patients in 6 families were diagnosed to have Mediterranean stomatocytosis/ macrothrombocytopenia. All had the presence of stomatocytes along with macrothrombocytopenia, short stature, continuous abdominal discomfort and marked pleiotropic effects in different cases. This is a syndromic form of stomatocytosis and defects in ABCG5/ABCG8 genes were found and showed an autosomal recessive inheritance pattern. One case did not show any mutation. This number of cases suggests that this disorder is not rare in India and is probably underdiagnosed as patients have mild or moderate anemia and are often misdiagnosed as cases with HS. One of the patients also had coinheritance of G6PD deficiency (G6PD Kerela Kalyan). Patients with Mediterranean stomatocytosis/macrothrombocytopenia are advised to take sterol-absorption inhibitor ‘ezetimibe’ to reduce sterol accumulation.We also found 2 unrelated patients with stomatocytosis, reticulocytosis and splenomegaly with overhydrated hereditary stomatocytosis (OHSt) and pathogenic mutation in RHAG gene was found in both of them. The pattern of inheritance is sporadic or autosomal dominant. Splenectomy was deferred in a patient with OHSt as postsplenectomy thrombotic complications are known to occur and is contraindicated.Four patients in 3 families were found to have mutations in PIEZO1 gene which translates to red cell membrane mechanosensitive cation channel protein, causing xerocytosis/dehydrated hereditary stomatocytosis and 3 patients had severe anemia and were transfusion dependent.One patient showed the presence of stomatocytes and macrothrombocytopenia was found to have probably disease causing variant in SLC2A1 gene. She was incidentally undergoing treatment for infertility when the stomatocytosis was noted.ConclusionsStomatocytic disorders appear to be underdiagnosed in India which is compounded by the protean clinical manifestations, milder phenotypes, low index of suspicion and non-availability of molecular confirmation. Astute phenotype characterization is critical as it will help in establishing the causality of the variants identified and appropriate genetic counseling. Recently NGS for hemolytic anemias has led to rapid molecular characterization and accurate phenotypic correlation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5091: Aneuploidy is in stomatin overexpressed cells

Abstract A 31.5 kDa membrane associated protein which is highly abundant in the lipid rafts is stomatin. Human gene expression profile from UCSC Genome Bioinformatics databank indicates that stomatin is major in the peripheral blood, adipose tissue, muscle, and bone marrow. In the beginning, stomatin was thought to be involved in the disease OHSt (overhydrated hereditary stomatocytosis), and therefore named stomatin. However, later studies did not reveal mechanistic role of stomatin in mediating OHSt by evaluating stomatin gene deleted mice; the function of stomatin is therefore remained unclear. In this study, we found that overexpression of stomatin was able to induce the multinucleated cell formation. Our data also demonstrated that stomatin made in the cytoplasm could be transported outside of the cell and released to the culture medium. While adding the culture medium or condition medium to the cells, which did not contain exogenous stomatin, these cells were also triggered to undergo formation of multinucleated cells. Additionally, the stomatin induced multinucleated cells presented aneuploidy and abnormal centrosome number. Possible mechanisms underlying functions of stomatin that induce multinucleated cells are investigated. Note: This abstract was not presented at the meeting. Citation Format: Jui-Hao Lee, Chi-Hung Lin. Aneuploidy is in stomatin overexpressed cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5091. doi:10.1158/1538-7445.AM2014-5091

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH(3) transport through the channel and NH(3) diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh(null) lacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

BAND 3CEINGE (Gly796Arg) Mutation Causes Dehydrated Hereditary Stomatocytosis (DHS) with Dyserythropoietic Phenotype

Stomatocytosis is an inherited autosomal dominant hemolytic anemia and includes overhydrated hereditary stomatocytosis (OHS), dehydrated hereditary stomatocytosis (DHS), hereditary cryohydrocytosis (CHC) and familial pseudohyperkalemia (FP). Here, we report a novel variant of hereditary stomatocytosis due to a de-novo band 3 mutation due to G>A transition at nucleotide 2500 in exon 17 (p. G796R, band3CEINGE) associated with dyserythropoietic phenotype. This 43-years-old Caucasian female (II-2) with unrelated parents was admitted to our hospital for mild anemia evaluation. The patient was in good health until 7 years when she frequently experienced asthenia. Anemia was first recognized at the age of eighth years with presence of jaundice and hyperchromic urine, but she had never received blood transfusions. We observed a mild hypochromic macrocytic anemia with a hemoglobin level of 11.5 g/dL, a mean cell volume (MCV) of 110 fL, and a mean hemoglobin concentration (MCH) of 36.1 pg, the reticulocyte count was 64 × 103/μL. There was a typical hemolytic features: high levels of indirect bilirubin (3.48 mg/dL) and lactate dehydrogenase ( 567 U/l, v.n. 240– 480 U/l ) with negativity at direct and indirect Coomb's test. Spleen was enlarged and ultrasonography detected 15 cm of longitudinal size. She was cholecystectomized at the age of 14 years because of numerous symptomatic small stones. Serum iron, soluble transferrin receptor, serum ferritin and transferrin saturation levels were all increased, while the transferrin was in the normal range.Other blood tests including osmotic fragility with incubated and fresh erythrocytes, serum electrolytes, B12 and folate levels, erythrocyte enzyme levels, EMA test and Pink test were normal. Peripheral blood smear showed anisopoikilocytosis with rare stomatocytes and no spherocytes. Bone marrow aspirate showed remarkable dyserythropoiesis with increased number of erythroblasts and binucleate erythroblasts, basophilic erythroblasts with alterations, irregular nuclei maturation, intererythroblastic bridges and erythroblasts with basophilic stippling. She received since the age of 14 yrs a diagnosis for congenital dyserythropoietic anemia type I. Patients red cells showedincrease Na+ content and decrease K+ content;reduced Na-K pump activity andincreased Na-H exchange, NKCC cotransport and KCC cotransport activities.We then functionally characterized band 3 CEINGE in Xenopus oocytes, showing that the mutated band 3 is converted from anion exchanger (Cl−, HCO3 −) function to unregulated cation pathway for Na+ and K+. The mutated band 3 was also associated with increased tyrosine phosphorylation pattern of some red cell membrane proteins. During erythropoiesis band 3 protein is the last cytoskeletal protein to appear, thus the dyserythropoietic phenotype may be related to a possible role of the mutated band 3 in perturbation of cytoskeleton assembly in the late stage of erythropoiesis, allowing us to conclude for a new variant of stomatocytosis with dyserythropoietic phenotype.

Erythrocyte Glut1 Triggers Dehydroascorbic Acid Uptake in Mammals Unable to Synthesize Vitamin C

Of all cells, human erythrocytes express the highest level of the Glut1 glucose transporter. However, the regulation and function of Glut1 during erythropoiesis are not known. Here, we report that glucose transport actually decreases during human erythropoiesis despite a >3-log increase in Glut1 transcripts. In contrast, Glut1-mediated transport of L-dehydroascorbic acid (DHA), an oxidized form of ascorbic acid (AA), is dramatically enhanced. We identified stomatin, an integral erythrocyte membrane protein, as regulating the switch from glucose to DHA transport. Notably though, we found that erythrocyte Glut1 and associated DHA uptake are unique traits of humans and the few other mammals that have lost the ability to synthesize AA from glucose. Accordingly, we show that mice, a species capable of synthesizing AA, express Glut4 but not Glut1 in mature erythrocytes. Thus, erythrocyte-specific coexpression of Glut1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.

Membrane raft actin deficiency and altered Ca 2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis

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 Ca 2+-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.

Stomatin is mis‐trafficked in the erythrocytes of overhydrated hereditary stomatocytosis, and is absent from normal primitive yolk sac‐derived erythrocytes

The 32 kD lipid-raft-associated membrane protein 'stomatin' is deficient from the erythrocyte membrane in the Na+-K+ leaky haemolytic anaemia, overhydrated hereditary stomatocytosis (OHSt). To date, no mutation in the gene coding for this protein has so far been found in OHSt. In this study, we have analysed the distribution of stomatin in both cultured erythroid cells from OHSt patients and in normal embryological and fetal erythroid development. In erythroid cell cultures from OHSt patients, stomatin-immunoreactivity (stomatin-IR) was present in progenitor cells but remained restricted to the area of the multivesicular complexes and the nucleus in the developing cells and was not seen in the plasma membrane. This could be consistent with the idea that stomatin is an innocent passenger in a more fundamental trafficking abnormality. In normal embryonic development, we found that, in extraembryonic (yolk sac) erythropoiesis, neither the nucleated red cells nor their enucleated mature derivatives displayed any stomatin-IR. In contrast, all haemangiopoietic progenitor cells of intraembryonic haematopoiesis, starting with the mesodermal precursors in the aorta-gonad-mesonephros region, exhibited strong stomatin-IR. The significance of this observation on these poorly understood cells is currently unclear.

Stomatocytosis of Standard Schnauzers is not associated with stomatin deficiency

Stomatocytosis resembles human overhydrated hereditary stomatocytosis (OHSt), a disease characterised by a reduced or absent stomatin expression. The objective of this report was to investigate the expression level of stomatin in erythrocytes from Standard Schnauzers with stomatocytosis. Routine haematology, intraerythrocytic Na +/K + concentration and stomatin expression were evaluated in blood from twelve Standard Schnauzers and from three controls. SDS–PAGE and Western blotting on isolated integral membrane proteins were used to investigate stomatin expression. Circulating stomatocytes, macrocytosis, anisocytosis, increased erythrocyte fragility and high intracellular sodium and potassium concentrations were found in 10/12 dogs from the same breeding line although stomatin levels were similar to those of controls. In spite of the clinico-pathological similarities between human and canine stomatocytosis, erythrocytes from affected dogs do not lack stomatin and the expression level of this protein cannot therefore be used to diagnose hereditary stomatocytosis in Standard Schnauzers.

The hereditary stomatocytoses: genetic disorders of the red cell membrane permeability to monovalent cations

The hereditary stomatocytoses are mostly accounted for by genetic disorders of red cell membrane permeability to monovalent cations. These conditions, all very rare, are comprised of a hemolytic anemia, frequently macrocytosis, and the presence of abnormally shaped red blood cells. The key test for diagnosis is osmotic gradient ektacytometry, which measures the osmotic resistance and hydration of the red blood cell; the curve depicting the temperature dependence of the cation leak is also important. Syndromes include familial pseudohyperkalemia (FP), which is devoid of hematological features, dehydrated hereditary stomatocytosis (DHS), and overhydrated hereditary stomatocytosis (OHS). Some forms of DHS may be a pleiotropic, showing pseudohyperkalemia and/or perinatal edema. Perinatal edema, if not properly treated, may be lethal but may also resolve spontaneously prior to or shortly after birth and never reappear. Hereditary cryohydrocytosis, type 1 (CHC 1) is characterized by a dramatic resumption of the leak in vitro as the temperature approaches 0°C; cell hydration seems unaltered. In OHS, stomatin, a membrane protein, is sharply reduced; however, this is a secondary event and the primarily mutated protein remains unknown. Hereditary cryohydrocytosis, type 2 (CHC 2) presents similar to OHS, except that the leak dramatically increases close to 0°C. In addition, hematological manifestations are associated with neurological disorders. Of critical practical importance is that splenectomy in DHS or OHS causes thromboembolic events that may be fatal. The genes involved in hereditary stomatocytoses have yet to be identified. Apart from the 16q24-qter locus, related to subsets of DHS and FP, and a chromosome 2 locus assigned to a single case of FP, gene mapping has been difficult. The eventual discovery of individual genes will clarify complicated classification of the stomatocytoses, now based solely on phenotype.

The “stomatin” gene and protein in overhydrated hereditary stomatocytosis

AbstractIn 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.