Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases; PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation, as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets.

Breath-holding spells occur disproportionately more often in children with transient erythroblastopenia.

AIM: This study aimed to evaluate the concomitant occurrence and possible association of breath-holding spells (BHS) and transient erythroblastopenia of childhood (TEC). METHODS: This population-based cohort study, carried out in Southern Sweden from 2004 to 2014, included patients with BHS and/or anaemia, including TEC. The subjects were evaluated for the presence of all three conditions and the diagnostic workups, disease characteristics and outcome were analysed. RESULTS: We studied 443 470 children under the age of 10 years during 2004-2014. The total cohort included 321 patients (0.07%) with BHS and 366 patients with a selection of anaemia diagnoses, including 41 with TEC. We found that nine (2.5%) of the 366 patients with anaemia diagnoses also had BHS and that five (12.2%) of the 41 patients with TEC also had BHS. Treatment for anaemia resolved BHS in a number of patients. CONCLUSION: Our population-based analysis revealed an overrepresentation of BHS among children with TEC, and we identified five patients with concomitant TEC and BHS. We found that correcting anaemia was an effective means of ameliorating potentially debilitating BHS and that the presence of concomitant BHS and TEC was more common than previously assumed.

Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.

Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant's loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P1/2, for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ∼6-20 nm(2) during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (∼200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.

[A case of acute opisthorchiasis concurrent with chronic hereditary hemolytic anemia].

The paper analyzes the clinical and laboratory manifestations of acute Opisthorchis invasion concurrent with chronic hereditary hemolytic anemia before and after antihelminthic therapy. It gives the results of direct clinical observation of a patient in the acute phase of opisthorchiasis in the presence of Minkowsky-Shauffard disease. His examination encompassed conventional laboratory and instrumental studies used in hepatology, such as physical, biochemical, and immunological examinations (tests for viral hepatitismarkers and autoimmune liver diseases), and abdominal ultrasound scanning and magnetic resonance imaging. The patient with acute opisthorchiasis concurrent with hemolytic anemia was found to have a preponderance of clinical and laboratory manifestations of hepatocholangitis in the early stages of the disease and a prevalence of subfebrility with progressive eosinophilia in the presence of regressive symptoms. The clinical and laboratory signs of hereditary microspherocytosis suggest that the process is decompensated. The found clinical and laboratory changes correspond to the natural course of the diseases. The magnitude of changes in laboratory parameters suggests that there is an intercurrent interaction of infectious and somatic diseases, but does not hinder dehelminthization.

Heme oxygenase-1 deficiency presenting with interstitial lung disease and hemophagocytic flares.

BACKGROUND: Heme oxygenase-1 (HMOX1) catalyzes the metabolism of heme into carbon monoxide, ferrous iron, and biliverdin. Through biliverdin reductase, biliverdin becomes bilirubin. HMOX1-deficiency is a rare autosomal recessive disorder with hallmark features of direct antibody negative hemolytic anemia with normal bilirubin, hyperinflammation and features similar to macrophage activation syndrome. Clinical findings have included asplenia, nephritis, hepatitis, and vasculitis. Pulmonary features and evaluation of the immune response have been limited. CASE PRESENTATION: We present a young boy who presented with chronic respiratory failure due to nonspecific interstitial pneumonia following a chronic history of infection-triggered recurrent hyperinflammatory flares. Episodes included hemolysis without hyperbilirubinemia, immunodeficiency, hepatomegaly with mild transaminitis, asplenia, leukocytosis, thrombocytosis, joint pain and features of macrophage activation with negative autoimmune serologies. Lung biopsy revealed cholesterol granulomas. He was found post-mortem by whole exome sequencing to have a compound heterozygous paternal frame shift a paternal frame shift HMOX1 c.264_269delCTGG (p.L89Sfs*24) and maternal splice donor HMOX1 (c.636 + 2 T > A) consistent with HMOX1 deficiency. Western blot analysis confirmed lack of HMOX1 protein upon oxidant stimulation of the patient cells. CONCLUSIONS: Here, we describe a phenotype expansion for HMOX1-deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares with hemophagocytosis present in the bone marrow.

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis.

BACKGROUND: Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. DESIGN AND METHODS: We report a novel variant of hereditary stomatocytosis due to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patient's erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events. RESULTS: The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na(+) content with decreased K(+)content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl(-), HCO(3)(-)) to being a cation pathway for Na(+) and K(+). Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient's red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events. CONCLUSIONS: Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis.

Total blood volume is maintained in nonhydropic fetuses with severe hemolytic anemia.

OBJECTIVE: Fetal alloimmune anemia is associated with increased blood flow velocities and cardiomegaly. In severe cases, hydrops can develop. We investigated whether the decrease of red blood cell volume is associated with a reduction or expansion of plasma volume. METHODS: In 86 alloimmunized fetuses that received a first intrauterine transfusion, we calculated fetal total blood volumes (i.e. fetoplacental blood volumes) using a dilutional principle of fetal hemoglobin with adult hemoglobin. The relation between total blood volume and estimated fetal weight, severity of anemia and hydrops was analyzed. RESULTS: Gestational age ranged from 17 to 35 weeks. Mean hemoglobin deficit was 6.8 standard deviations (range 2.1-11.7) below the normal mean. Fetal total blood volume was significantly related to estimated fetal weight (p < 0.001). Mean total blood volume in nonhydropic fetuses was 123 ml/kg (n = 74) and in hydropic fetuses 144 ml/kg (n = 12). There was a significant relation between total blood volume per kg body weight and hydrops (p = 0.035); however, there was no relation with severity of anemia (p = 0.94). CONCLUSION: In the human nonhydropic fetus with severe hemolytic anemia, total blood volume is maintained: the decrease in red blood cell volume is thus compensated by an increase in plasma volume. In hydropic fetuses, however, total blood volume seems to be increased. This is in accordance with the hypothesis that congestive heart failure plays a role in the pathophysiology of hydrops in anemic fetuses.

[Hereditary xerocytosis. Presentation of two pediatric cases]. / Xerocitosis hereditaria. Presentación de dos casos clínicos pediátricos.

Hereditary xerocytosis is a rare disorder caused by defects of red blood cell permeability that are characterized by hemolytic anemia of variable degree and iron overload. Diagnosis is usually late and confused with other hemolytic anemias, which can lead to procedural indications, such as splenectomy, contraindicated in these patients. We report the clinical, haematological, and molecular characteristics of two patients from two unrelated families affected by hereditary xerocytosis. Both patients had dehydrated erythrocytes with a high concentration of mean corpuscular hemoglobin, non-pathognomonic smears, markers of hemolysis and a resistant osmotic fragility curve. The diagnosis was confirmed by the sequencing of the PIEZO gene. We emphasize the importance of recognizing the cause of hemolytic anemia to give an accurate therapeutic approach and give adequate genetic counseling.

La xerocitosis hereditaria es un desorden poco frecuente causado por defectos en la permeabilidad eritrocitaria, que se caracteriza por anemia hemolítica de gravedad variable y sobrecarga de hierro. El diagnóstico suele ser tardío y confundirse con otras anemias hemolíticas, lo que puede llevar a indicaciones de procedimientos, como la esplenectomía, contraindicados en estos pacientes. Se reportan las características clínicas, hematológicas y moleculares de dos pacientes pediátricos no relacionados con diagnóstico de xerocitosis hereditaria. Ambos presentaban eritrocitos deshidratados con alta concentración de hemoglobina corpuscular media, frotis no patognomónico, marcadores de hemólisis y una curva de fragilidad osmótica resistente. El diagnóstico se confirmó por la secuenciación del gen PIEZO. Se resalta la importancia de reconocer la causa de la anemia hemolítica para dar un enfoque terapéutico preciso y dar adecuado consejo genético.

Disorders of red cell membrane.

Studies during the last three decades have enabled the development of detailed molecular insights into the structural basis of altered function in various inherited red cell membrane disorders. This review highlights our current understanding of molecular and mechanistic insights into various inherited red cell membrane disorders involving either altered membrane structural organization (hereditary spherocytosis, hereditary elliptocytosis and hereditary ovalocytosis) or altered membrane transport function (hereditary stomatocytosis). The molecular basis for the vast majority of cases of hereditary spherocytosis, elliptocytosis and ovalocytosis have been fully defined while little progress has been made in defining the molecular basis for hereditary stomatocytosis. Mutations in a number of distinct genes account for hereditary spherocytosis and elliptocytosis, while a single genetic defect accounts for all cases of hereditary ovalocytosis. Based on these molecular insights, a comprehensive understanding of the structural basis for altered membrane function has been developed. Loss of vertical linkage between membrane skeleton and lipid bilayer leads to membrane loss in hereditary spherocytosis, while weakening of lateral linkages between skeletal proteins leads to membrane fragmentation and surface area loss in hereditary elliptocytosis. Importantly, the severity of anaemia in both these disorders is directly related to extent of membrane surface area loss. Splenectomy results in amelioration of anaemia.

The molecular basis of hereditary red cell membrane disorders.

The red cell membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any plasma membrane it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. Many of the proteins and the genes performing these functions are known in great detail, although some disease-responsible genes are yet to be elucidated. Basic knowledge has shed light on important groups of genetic disorders. The latter include (i) the disorders of the red cell mechanics: hereditary spherocytosis, hereditary elliptocytosis and poikilocytosis, and (ii) the disorders of the passive flux of the monovalent cations across the membrane: the stomacytoses and allied conditions. Reciprocally, many information have come from genetics abnormalities. We will review the mutation-disease relationship. A number of points will be underscored: widespread weak alleles modulate the expression of the SPTA1 gene, encoding the alpha-chain of spectrin; mutations in the anion exchanger can give rise to an array of distinct nosological entities, including a renal condition; splenectomy is banned in the stomatocytoses; a variety of stomatocyosis is part of a pleiotropic syndrome that may includes perinatal fetal liquid effusions. The diagnosis, follow-up and treatment of the involved diseases have gradually improved.

Studies on the pathogenesis of pigment gallstones in hemolytic anemia: description and characteristics of a mouse model.

The pathogenesis of hemolysis-induced gallstones was studied in mice with a hereditary hemolytic disease called normoblastic anemia (genotype nb/nb) and in their normal controls (genotype +/+). Infrared spectroscopy demonstrated that spontaneously formed gallstones from nb/nb mice were nearly identical to stones from patients with chronic hemolysis as the result of sickle cell disease, and both mouse and human stones strikingly resembled synthetic calcium bilirubinate. 57% of 115 nb/nb mice, but none of 109 control mice, developed calcium bilirubinate pigment gallstones (P < 0.001). The incidence of luminal gallstones in nb/nb mice was both sex and age dependent. Female nb/nb mice formed stones twice as frequently as male nb/nb mice (P < 0.001). Before 6 mo of age neither sex developed stones, but thereafter the incidence of stones increased with age. Neither hematocrit, reticulocyte count, nor total plasma bilirubin values, were useful in distinguishing between nb/nb mice with or without gallstones. In gallbladder bile, nb/nb mice with gallstones had higher concentrations of hydrogen ion, total bilirubin, calcium, and bile acids than nb/nb mice without stones. Although total unconjugated bilirubin was similar in both nb/nb groups, the ionized fraction of unconjugated bilirubin was higher in bile from nb/nb mice without stones than those with stones. In nb/nb mice, neutral mucin plugs and pigment concentrations were observed histologically in the glandular crypts of the gallbladder in 33% of nb/nb mice without stones and in 80% of nb/nb mice with luminal stones. This suggested that luminal pigment stone disease in mice with hemolysis may be preceded by microscopic precipitation of calcium bilirubinate in the glandular crypts of the gallbladder. These precipitates may then migrate into the lumen and grow by accretion.

Congenital Hemolytic Anemia.

Red blood cell (RBC) destruction can be secondary to intrinsic disorders of the RBC or to extrinsic causes. In the congenital hemolytic anemias, intrinsic RBC enzyme, RBC membrane, and hemoglobin disorders result in hemolysis. The typical clinical presentation is a patient with pallor, anemia, jaundice, and often splenomegaly. The laboratory features include anemia, hyperbilirubinemia, and reticulocytosis. For some congenital hemolytic anemias, splenectomy is curative. However, in other diseases, avoidance of drugs and toxins is the best therapy. Supportive care with transfusions are also mainstays of therapy. Chronic hemolysis often results in the formation of gallstones, and cholecystectomy is often indicated.

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton.

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.

Hemopoietic support capacity of W/WV femurs and tibias.

We evaluated the capacity of "stromal stem cells" of the bones of W/WV mice to effect regeneration of functional "stromal" tissue by implanting femurs and tibias from congenic W/WV and +/+ mice subcutaneously into congenic mice of normal hematologic phenotypes. Eight weeks after implantation, we assayed the hemopoietic progenitor cell contents of the bones. Pluriopotent hemopoietic stem cells (CFUs) were assayed by the spleen colony forming assay in lethally irradiated CAF1 mice. Granulocyte/macrophage progenitor cells (CFUGM) and early erythroid (BFUe) and late erythroid (CFUE) progenitor cells were assayed in agar and methylcellulose semi-solid culture systems, respectively. total cellularity was greater in implanted W/WV femurs and tibias than in +/+ femurs. Although there was substantial variation in the repopulation of W/WV femurs compared to that of +/+ femurs, on the whole, in the W/WV implanted femurs, the numbers of CFUs may have been slightly less and the numbers of maturer progenitor cells slightly greater than they were in the +/+ femurs. All of the progenitor cells assayed were more numerous in the implanted W/WV tibias than in the +/+ tibias. These findings suggest that "stromal stem cells" of W/WV marrow are not defective. In addition, the fact that there were more CFUs in th W/WV that in the +/+ tibias suggests that the CFUs which are native to the implanted bones are not needed to effect reconstitution of normal numbers of CFUs in the implants as all of the CFUs detected in W/WV femurs must have come from the host, for W/WV CFUs do not form surface spleen colonies and, therefore, would not have been counted.

In vitro hemopoiesis of W/WV and +/+ marrow cells cultured alone and with thymocytes.

To evaluate the effects of thymocytes from anemic W/WV mice on hemopoiesis in vitro, we co-cultured thymocytes from W/WV mice or from their non-anemic +/+ littermates with W/WV or +/+ marrow cells in semi-solid systems designed to foster colonial growth from neutrophil/macrophage (CFUNM), early erythroid (BFUE), late erythroid (CFUE) precursors. The marrow cell to thymocyte ratios in the cultures were 1:3 and 1:20 (1:175 for CFUNM). Untreated or anti-theta serum (ATS) or mock (control) AT treated marrow cells were cultured with the thymocytes. We found that marrow from +/+ and W/WV mice contain equivalent concentrations of CFUNM but W/WV marrow has a lower concentration of BFUE than +/+ marrow. Neither +/+ nor W/WV thymoctyes, in the concentrations used, effected any increase in the numbers of CFUNM or CFUE colonies formed from untampered or antiserum treated marrow cells. In two groups, thymocytes effected a slight increase in the numbers of bursts (BFUE colonies) formed from marrow cell suspensions and in both of these groups W/WV thymocytes were as effective as +/+ thymocytes. Thus, under the conditions studied, W/WV thymocytes were not more inhibitory or less stimulatory for in vitro hemopoiesis than were normal thymocytes.

Spectrin: structure, function, and abnormalities.

A number of proteins that make up the membrane skeleton have been identified, and we have a rough but tantalizing picture of the ways in which they interact to maintain its integrity. An approximate molecular model of spectrin has been proposed, and many new analytic procedures have been developed to search for biochemical variants that may be related to membrane defects. Even at this rudimentary stage of molecular description we have been able to identify structural changes in spectrin that are correlated with pathophysiologic states. The prospects for some genuine insights into the pathogenesis of some congenital hemolytic anemias seem good.

Clinical features and splenic pathologic changes in patients with autoimmune hemolytic anemia and congenital hemolytic anemia.

We characterized the pathologic changes in 50 spleens from patients with autoimmune hemolytic anemia and in 13 spleens from patients with congenital hemolytic anemia. The major pathologic findings in autoimmune hemolytic anemia were mild to pronounced cord congestion and variable white pulp area, erythrophagocytosis in conjunction with an increased polymorphonuclear neutrophil reaction, and increased deposition of hemosiderin and extramedullary hematopoiesis. In contrast, both the severity and the frequency of polymorphonuclear neutrophil reactions, deposition of hemosiderin, and extramedullary hematopoiesis were less in patients with congenital hemolytic anemia, and prominent cord congestion associated with an empty or collapsed sinus was noted in patients with hereditary spherocytosis. The activity of erythrophagocytosis in the sinus was more distinct in patients with congenital hemolytic anemia, especially those with nonspherocytic congenital hemolytic anemia, than in patients with autoimmune hemolytic anemia. These findings are consistent with the existence of different mechanisms of trapping and destruction of erythrocytes in autoimmune hemolytic anemia and congenital hemolytic anemia.