Southeast Asian Ovalocytosis and Hemoglobinopathies in Newborns: Prevalence, Molecular, and Hematologic Analyses.

OBJECTIVES: Southeast Asian ovalocytosis (SAO) is an inherited red blood cell (RBC) membrane disorder, whereas hemoglobinopathies are inherited globin gene disorders. In an area where both diseases are prevalent, the interaction between them resulting in variable hematologic parameters can be encountered. However, little is known about the genetic interaction of SAO and thalassemia. We investigated the prevalence of SAO and hemoglobinopathy genotypes among newborns in southern Thailand. PATIENTS AND METHODS: This study was carried out on 297 newborns recruited consecutively at Naradhiwas Rajanagarindra Hospital in the south of Thailand. The SAO was identified on blood smear examination and polymerase chain reaction analysis. Thalassemia genotypes were defined. Hematologic parameters and hemoglobin (Hb) profiles were recorded and analyzed. RESULTS: Among 297 newborns, 15 (5.1%) carried SAO, whereas 70 (23.6%) had thalassemia with 15 different thalassemia genotypes. Abnormal Hb including Hb C, Hb Q-Thailand, and Hb D-Punjab were observed in 5 newborns. It was found in the nonthalassemic newborns that RBC count, Hb, and hematocrit of the nonthalassemic newborns with SAO were significantly lower than those without SAO. The same finding was also observed in the thalassemic newborns; RBC count, Hb, and hematocrit of the thalassemic newborns with SAO were significantly lower than those without SAO. However, the mean corpuscular volume, mean corpuscular Hb, and RBC distribution width of the SAO-newborns were significantly higher. CONCLUSIONS: Both SAO and hemoglobinopathy genotypes are common in southern Thailand. One should take this into consideration when evaluating neonatal anemia and other hematologic abnormalities. Identification of both genetic defects and long-term monitoring on the clinical outcome of this genetic interaction should be essential to understand the pathogenesis of these common genetic disorders in the region.

A case series of distal renal tubular acidosis, Southeast Asian ovalocytosis and metabolic bone disease.

BACKGROUND: Familial distal renal tubular acidosis (dRTA) associated with mutations of solute carrier family 4 membrane - 1 (SLC4A1) gene could co-exist with red cell membrane abnormality, Southeast Asian ovalocytosis (SAO). Although this association is well described in Southeast Asian countries, it is less frequently found in Sri Lanka. CASE PRESENTATION: We describe six patients who had dRTA co-existing with SAO. All of them initially presented with severe hypokalemia and paralysis. They presented within a period of six months to the Teaching Hospital Anuradhapura, Sri Lanka. All had metabolic acidosis indicated by low serum bicarbonate. Three of them were having underlying chronic kidney disease as well. Those three patients had mixed high and normal anion gap metabolic acidosis indicated by low delta ratio. In all dRTA was confirmed by presence of normal anion gap, hyperchloraemia, high urine pH and positive urine anion gap. Examination of blood films of all of them revealed presence of stomatocytes and macro-ovalocytosis compatible with SAO. In relation to complications of dRTA, two patients had medullary nephrocalcinosis. Three patients had biochemical evidence of osteomalacia, with two of them having radiological evidence of diffuse osteosclerosis. One patient had secondary hyperparathyroidism and a pathological fracture. CONCLUSIONS: Erythrocyte in SAO is exceptionally rigid and this abnormality is said to be evolved as it protects against Plasmodium vivax malaria and cerebral malaria cause by Plasmodium falciparum. Although two families of SAO was described earlier, SAO and dRTA combination was reported only once in a patient from Anuradhapura district. Distal renal tubular acidosis, SAO combination and its related complications including nephrocalcinosis, chronic kidney disease and metabolic bone disease was not described in Sri-Lankan literature. This case series emphasize the importance of investigating recurrent/ chronic hypokalemia to diagnose dRTA and its associations, as early correction of acidosis could prevent development of chronic kidney disease and metabolic bone disease.

Dizygotic twins with prolonged jaundice and microcytic, hypochromic, hemolytic anemia with pyropoikilocytosis.

Dizygotic twin males, born at 34 weeks gestation, had prolonged jaundice, microcytic, hypochromic anemia, FABarts hemoglobin, elevated end-tidal CO, and blood films consistent with hereditary pyropoikilocytosis. DNA sequencing revealed both had a heterozygous alpha spectrin (SPTA1) mutation (c.460_462dup) inherited from their asymptomatic mother, plus a 3-base pair duplication in alpha globin (HBA2) (c.364_366dupGTG) inherited from their asymptomatic father.

Development of a novel test for the identification of hereditary erythrocyte membrane defects by TGA/Chemometrics.

Systematic screening for congenital erythrocyte disorders is not a common practice, due to a lack in the accuracy of the methods and to the costs of the analyses. As a consequence, the diagnosis is usually made when a severe complication occurs. This study introduces an innovative method to perform the screening of a hereditary disease characterized by erythrocyte membrane defects such as hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). Blood samples from healthy donors and patients affected by HS and HE were processed by thermogravimetric analysis (TGA) and the resulting curves were analyzed by chemometrics in order to develop a model of prediction. A novel test was developed and validated by chemometrics and results were found to be in accordance with the genetic diagnosis, confirming the prediction ability of the optimized model that uses few microliters of whole non pretreated blood to perform the diagnosis of HS/HE in less than one hour.

Advances in understanding the pathogenesis of red cell membrane disorders.

Hereditary erythrocyte membrane disorders are caused by mutations in genes encoding various transmembrane or cytoskeletal proteins of red blood cells. The main consequences of these genetic alterations are decreased cell deformability and shortened erythrocyte survival. Red blood cell membrane defects encompass a heterogeneous group of haemolytic anaemias caused by either (i) altered membrane structural organisation (hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and Southeast Asian ovalocytosis) or (ii) altered membrane transport function (overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis or xerocytosis, familial pseudohyperkalaemia and cryohydrocytosis). Herein we provide a comprehensive review of the recent literature on the molecular genetics of erythrocyte membrane defects and their reported clinical consequences. We also describe the effect of low-expression genetic variants on the high inter- and intra-familial phenotype variability of erythrocyte structural defects.

Mechanics of diseased red blood cells in human spleen and consequences for hereditary blood disorders.

In red blood cell (RBC) diseases, the spleen contributes to anemia by clearing the damaged RBCs, but its unique ability to mechanically challenge RBCs also poses the risk of inducing other pathogenic effects. We have analyzed RBCs in hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), two typical examples of blood disorders that result in membrane protein defects in RBCs. We use a two-component protein-scale RBC model to simulate the traversal of the interendothelial slit (IES) in the human spleen, a stringent biomechanical challenge on healthy and diseased RBCs that cannot be directly observed in vivo. In HS, our results confirm that the RBC loses surface due to weakened cohesion between the lipid bilayer and the cytoskeleton and reveal that surface loss may result from vesiculation of the RBC as it crosses IES. In HE, traversing IES induces sustained elongation of the RBC with impaired elasticity and fragmentation in severe disease. Our simulations thus suggest that in inherited RBC disorders, the spleen not only filters out pathological RBCs but also directly contributes to RBC alterations. These results provide a mechanistic rationale for different clinical outcomes documented following splenectomy in HS patients with spectrin-deficient and ankyrin-deficient RBCs and offer insights into the pathogenic role of human spleen in RBC diseases.

Accurate light microscopic diagnosis of South-East Asian ovalocytosis.

INTRODUCTION: South-East Asian ovalocytosis (SAO) is a common inherited red blood cell polymorphism in South-East Asian and Melanesian populations, coinciding with areas of malaria endemicity. Validation of light microscopy as a diagnostic alternative to molecular genotyping may allow for its cost-effective use either prospectively or retrospectively by analysis of archived blood smears. METHODS: We assessed light microscopic diagnosis of SAO compared to standard PCR genotyping. Three trained microscopists each assessed the same 971 Giemsa-stained thin blood films for which SAO genotypic confirmation was available by PCR. Generalized mixed modeling was used to estimate the sensitivity, specificity, positive predictive value, and negative predictive value of light microscopy vs "gold standard" PCR. RESULTS: Among red cell morphologic parameters evaluated, knizocytes, rather than ovalocytic morphology, proved the strongest predictor of SAO status (odds ratio [OR] = 19.2; 95% confidence interval [95% CI] = 14.6-25.3; P ≤ 0.0001). The diagnostic performance of a knizocyte-centric microscopic approach was microscopist dependent: two microscopists applied this approach with a sensitivity of 0.89 and a specificity of 0.93. Inter-rater reliability among the microscopists (κ = 0.20) as well as between gold standard and microscopist (κ = 0.36) underperformed due to misclassification of stomatocytes as knizocytes by one microscopist, but improved substantially when excluding the error-prone reader (κ = 0.65 and 0.74, respectively). CONCLUSION: Light microscopic diagnosis of SAO by knizocyte visual cue performed comparable to time-consuming and costlier molecular methods, but requires specific training that includes successful differentiation of knizocytes from stomatocytes.

Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis.

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin­5­maleimide (EMA)­labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix­assisted laser desorption­ionization time of flight (MALDI­TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large­scale gene screening using high­resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS­PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA­labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531­12C>T. The corresponding mutant polypeptides were also analyzed by MALDI­TOF mass spectroscopy. SDS­PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression.

Southeast asian ovalocytosis: the need for a carefull observation of red cell indices and blood smear.

Southeast asian ovalocytosis (SAO) is characterized by macro-ovalocytes and ovalo-stomatocytes on blood smear. SAO is common in Malaisia and Papua-New-Guinea where upwards to 40 per cent of the population is affected in some coastal region. Inherited in an autosomal dominant way, illness results from deletion of codons 400-408 in SLC4A1 gene which encodes for band 3 erythrocyte membrane protein. This deletion is responsible for an unusual erythrocyte stiffness and oval shape of the cells on blood smear. Heterozygous carriers are usually asymptomatic whereas homozygous are not viable without an intensive antenatal care. Here, we describe 4 patients diagnosed incidentally by cytogram appearance of the Advia® 2120i (Siemens) representing hemoglobin concentration according to red blood mean cellular volume (GR/VCH).

Band 3, the human red cell chloride/bicarbonate anion exchanger (AE1, SLC4A1), in a structural context.

The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.

Modeling of band-3 protein diffusion in the normal and defective red blood cell membrane.

We employ a two-component red blood cell (RBC) membrane model to simulate lateral diffusion of band-3 proteins in the normal RBC and in the RBC with defective membrane proteins. The defects reduce the connectivity between the lipid bilayer and the membrane skeleton (vertical connectivity), or the connectivity of the membrane skeleton itself (horizontal connectivity), and are associated with the blood disorders of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) respectively. Initially, we demonstrate that the cytoskeleton limits band-3 lateral mobility by measuring the band-3 macroscopic diffusion coefficients in the normal RBC membrane and in a lipid bilayer without the cytoskeleton. Then, we study band-3 diffusion in the defective RBC membrane and quantify the relation between band-3 diffusion coefficients and percentage of protein defects in HE RBCs. In addition, we illustrate that at low spectrin network connectivity (horizontal connectivity) band-3 subdiffusion can be approximated as anomalous diffusion, while at high horizontal connectivity band-3 diffusion is characterized as confined diffusion. Our simulations show that the band-3 anomalous diffusion exponent depends on the percentage of protein defects in the membrane cytoskeleton. We also confirm that the introduction of attraction between the lipid bilayer and the spectrin network reduces band-3 diffusion, but we show that this reduction is lower than predicted by the percolation theory. Furthermore, we predict that the attractive force between the spectrin filament and the lipid bilayer is at least 20 times smaller than the binding forces at band-3 and glycophorin C, the two major membrane binding sites. Finally, we explore diffusion of band-3 particles in the RBC membrane with defects related to vertical connectivity. We demonstrate that in this case band-3 diffusion can be approximated as confined diffusion for all attraction levels between the spectrin network and the lipid bilayer. By comparing the diffusion coefficients measured in horizontal vs. vertical defects, we conclude that band-3 mobility is primarily controlled by the horizontal connectivity.

Characterization of iron metabolism and erythropoiesis in erythrocyte membrane defects and thalassemia traits.

BACKGROUND AND AIMS: Erythropoiesis is closely related to iron metabolism in a balanced homeostasis. Analyses of diverse erythroid and iron metabolism disorders have shown that disrupted erythropoiesis negatively affects iron homeostasis and vice versa. The aim of this study was to characterize the relationship between erythropoietic activity and iron homeostasis in pediatric patients with erythrocyte membrane defects and thalassemia traits. METHODS: Selected markers of erythropoietic activity (erythropoietin, soluble transferrin receptor - sTfR and growth differentiation factor 15) and iron status parameters (serum iron, ferritin and hepcidin) were evaluated in pediatric patients with erythrocyte membrane defects and thalassemia traits. RESULTS: The patients with erythrocyte membrane defects and thalassemia traits had altered iron homeostasis due to disturbed erythropoiesis. In comparison with healthy controls, they had a normal to low hepcidin/ferritin ratio and concomitantly elevated sTfR. CONCLUSION: The findings suggest that pediatric patients with erythrocyte membrane defects and thalassemia traits are more susceptible to iron overload than the general population and that the (hepcidin/ferritin)/sTfR ratio can be used to monitor any worsening of the disease.

[Erythrocyte membrane abnormalities - hereditary elliptocytosis].

This study was designed to investigate the 4 year old boy with Hereditary Elliptocitosis (HE). The diagnosis of this rare hemolytic anemia was based on detailed family history (positive in the 4-th generation), physical examination and Para-clinical data analyses. The vast majority of patients with HE are asymptomatic, severe forms are rare. The most important is examination of blood films, which is helpful to detect the morphology abnormalities of red cells. In case of HE a different approach is required. Positive family history and series of investigations should be conducted to determine the HE.