Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Minor Changes in Erythrocyte Osmotic Fragility in Trace Amine-Associated Receptor 5 (TAAR5) Knockout Mice.

Trace amine-associated receptors (TAARs) are a group of G protein-coupled receptors that are expressed in the olfactory epithelium, central nervous system, and periphery. TAAR family generally consists of nine types of receptors (TAAR1-9), which can detect biogenic amines. During the last 5 years, the TAAR5 receptor became one of the most intriguing receptors in this subfamily. Recent studies revealed that TAAR5 is involved not only in sensing socially relevant odors but also in the regulation of dopamine and serotonin transmission, emotional regulation, and adult neurogenesis by providing significant input from the olfactory system to the limbic brain areas. Such results indicate that future antagonistic TAAR5-based therapies may have high pharmacological potential in the field of neuropsychiatric disorders. TAAR5 is known to be expressed in leucocytes as well. To evaluate potential hematological side effects of such future treatments we analyzed several hematological parameters in mice lacking TAAR5. In these mutants, we observed minor but significant changes in the osmotic fragility test of erythrocytes and hematocrit levels. At the same time, analysis of other parameters including complete blood count and reticulocyte levels showed no significant alterations in TAAR5 knockout mice. Thus, TAAR5 gene knockout leads to minor negative changes in the erythropoiesis or eryptosis processes, and further research in that field is needed. The impact of TAAR5 deficiency on other hematological parameters seems minimal. Such negative, albeit minor, effects of TAAR5 deficiency should be taken into account during future TAAR5-based therapy development.

Genetic Deletion of Trace-Amine Associated Receptor 9 (TAAR9) in Rats Leads to Decreased Blood Cholesterol Levels.

In the last two decades, interest has grown significantly in the investigation of the role of trace amines and their receptors in mammalian physiology and pathology. Trace amine-associated receptor 9 (TAAR9) is one of the least studied members of this receptor family with unidentified endogenous ligands and an unknown role in the central nervous system and periphery. In this study, we generated two new TAAR9 knockout (TAAR9-KO) rat strains by CRISPR-Cas9 technology as in vivo models to evaluate the role of TAAR9 in mammalian physiology. In these mutant rats, we performed a comparative analysis of a number of hematological and biochemical parameters in the blood. Particularly, we carried out a complete blood count, erythrocyte osmotic fragility test, and screening of a panel of basic biochemical parameters. No significant alterations in any of the hematological and most biochemical parameters were found between mutant and WT rats. However, biochemical studies revealed a significant decrease in total and low-density lipoprotein cholesterol levels in the blood of both strains of TAAR9-KO rats. Such role of TAAR9 in cholesterol regulation not only brings a new understanding of mechanisms and biological pathways of lipid exchange but also provides a new potential drug target for disorders involving cholesterol-related pathology, such as atherosclerosis.

Characterization of hereditary red blood cell membranopathies using combined targeted next-generation sequencing and osmotic gradient ektacytometry.

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte.

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.

Ankyrin-1 Gene Exhibits Allelic Heterogeneity in Conferring Protection Against Malaria.

Allelic heterogeneity is a common phenomenon where a gene exhibits a different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host-parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified two Ank-1 mutations, one resulting in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1(MRI96570) mutation was found to inhibit intraerythrocytic parasite maturation, whereas Ank-1(MRI95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomena in achieving a better understanding of host-parasite interactions, which could be the basis of future studies.

Previously undiagnosed hereditary spherocytosis in a patient with jaundice and pyelonephritis: a case report.

BACKGROUND: Hereditary spherocytosis is autosomal dominant inherited extravascular hemolytic disorder and is the commonest cause of inherited hemolysis in northern Europe and the United States. The classical clinical features of hereditary spherocytosis are anemia, jaundice, and splenomegaly. However, all of these classical features are not always revealed in the case of mild hemolysis or when hemolysis is well compensated. Patients with hereditary spherocytosis may remain undiagnosed for years if their hemolysis is mild. CASE PRESENTATION: A 42-year-old Asian woman presented to our clinic with a sudden onset of high fever with shaking chills and jaundice, suggesting septicemia; however, following detailed investigation, the patient was diagnosed with pyelonephritis and accelerated hemolysis of hereditary spherocytosis due to infection. CONCLUSIONS: It is important to note that transient anemia or jaundice can sometimes be the only initial presenting symptoms in cases of undiagnosed latent hereditary spherocytosis. This case also highlights the fact that physicians should consider concomitant hemolytic disease in patients in whom jaundice and infections that rarely cause jaundice coexist.

Recomendación del uso de ecuaciones de corrección de valores de potasio en presencia de interferencia por hemólisis / Recommendation on the use of correction equations for potassium levels in haemolysed samples

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Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice.

BACKGROUND: Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. METHODS: We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FINDINGS: Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. INTERPRETATION: The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

Erythroid transcription factor EKLF/KLF1 mutation causing congenital dyserythropoietic anemia type IV in a patient of Taiwanese origin: review of all reported cases and development of a clinical diagnostic paradigm.

KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.

Macrothrombocytopenia/stomatocytosis specially associated with phytosterolemia.

Phytosterolemia is a rare autosomal recessive disease of plant sterol metabolism, the pathophysiological features of which are high plasma levels of plant sterols and xanthomatosis caused by mutations of ABCG5 and ABCG8 genes, and the combination of hemolysis and macrothrombocytopenia is an unusual clinical manifestation. All the patients of the 3 unrelated phytosterolemia first presented with prominent macrothrombocytopenia and stomatocytosis. They were either homozygous or compound heterozygous for ABCG5/ABCG8 gene mutations and had significantly elevated serum plant sterols levels quantified using high-performance liquid chromatography. The in vitro study demonstrated that sitosterol can cause changes in shape and osmotic fragility of red blood cells. These findings suggest that macrothrombocytopenia and stomatocytosis could be initial and main features in some patients with phytosterolemia and that serum phytosterols and relevant genes should be analyzed in patients whose macrothrombocytopenia and/or stomatocytosis are unexplained, especially whose parents are of consanguineous marriage.

Comparative proteomics reveals deficiency of NHE-1 (Slc9a1) in RBCs from the beta-adducin knockout mouse model of hemolytic anemia.

Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.

Targeted deletion of alpha-adducin results in absent beta- and gamma-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice.

In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

Combined deletion of mouse dematin-headpiece and beta-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia.

Dematin and adducin are actin-binding proteins of the erythrocyte "junctional complex." Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking beta-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an approximately 12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.

Mild dehydrated hereditary stomatocytosis revealed by marked hepatosiderosis.

We report a patient in whom hepatosiderosis was diagnosed at the age of 55 years and who has since been treated by regular bleeding. The H63D mutation was found in the heterozygous state in the HFE gene. No mutation was recorded in the SLC11A3 gene (ferroportin). Hepatosiderosis did not seem primary, nevertheless its cause long remained elusive. Only 2 years ago did we find the responsible condition, a very mildly expressed form of dehydrated hereditary stomatocytosis (DHS). This genetic disease is a strongly iron-loading condition. Haemolysis was fully compensated. Kalaemia was slightly elevated, suggesting a pseudohyperkalaemia that may be associated with DHS. Osmotic gradient ektacytometry allowed to assess the diagnosis of DHS. The red cell monovalent Na+ and K+ concentrations were moderately elevated and reduced respectively. The temperature dependence of the ouabain + bumetanide-resistant K+ influx produced a shallow slope, above and parallel to the control curve. These features were consistent with the diagnosis of DHS. The pronounced hepatosiderosis contrasted with the mildly expressed DHS, and with the ferritinaemia that was slightly elevated, if at all, prior to bleeding. Bleeding caused ferritinaemia to decrease and hepatosiderosis to recede. The whole picture accounts for a misleading presentation of DHS, in which the primary condition long remained hidden behind one of its remotest complications, hepatosiderosis.

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.

To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.

The kinetics of haemolysis of spherocytic erythrocytes.

Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.

Putative homologs of SSK22 MAPKK kinase and PBS2 MAPK kinase of Saccharomyces cerevisiae encoded by os-4 and os-5 genes for osmotic sensitivity and fungicide resistance in Neurospora crassa.

We cloned and characterized Neurospora NcSSK22 and NcPBS2 genes, similar to yeast SSK22 mitogen-activated protein (MAP) kinase kinase kinase and the PBS2 MAP kinase kinase genes, respectively. Disruptants of the NcSSK22 gene were sensitive to osmotic stress and resistant to iprodione and fludioxonil. Their phenotypes were similar to those of osmotic-sensitive (os) mutants os-1, os-2, os-4, and os-5. The os-4 mutant strain transformed with the wild-type NcSSK22 gene grew on a medium containing 4% NaCl and was sensitive to iprodione and fludioxonil. In contrast, the NcPBS2 gene complemented the osmotic sensitivity and fungicide resistance of the os-5 mutant strain. We sequenced the NcPBS2 gene of the os-5 mutant strain (NM216o) and found five nucleotides deleted within the kinase domain. This result suggests that the gene products of os-4 and os-5 are components of the MAP kinase cascade, which is probably regulated upstream by two-component histidine kinase encoded by the os-1/nik1 gene.

Haemoglobin Q-Thailand and hereditary spherocytosis in a Chinese family.

A Chinese family with concurrent hereditary spherocytosis (HS) and haemoglobin (Hb) Q-Thailand is described. The Hb Q-Thailand mutation was found on the remaining alpha1 globin gene on a chromosome 16 containing the (-alpha 4.2) deletion. Active haemolysis in members of this family is segregated with the HS phenotype, and the Hb Q-Thailand in the heterozygous state does not seem to show any modulating effect on HS.

Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice.

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.