Distal Renal Tubular Acidosis in an Iranian Patient with Hereditary Spherocytosis

Background: Hereditary spherocytosis (HS) and hereditary hereditary distal renal tubular acidosis (dRTA) are associated with mutations in the SLC4A1 gene encoding the anion exchanger 1. In this study, some patients with clinical evidence of congenital HS and renal symptoms were investigated. Methods: Twelve patients with congenital HS and renal symptoms were recruited from Ali-Asghar Children's Hospital (Tehran, Iran). A patient suspected of having dRTA was examined using whole exome sequencing method, followed by Sanger sequencing. Results: One patient (HS03) showed severe failure to thrive, short stature, frequent urinary infection, and weakness. A homozygote (rs571376371 for c.2494C>T; p.Arg832Cys) and a heterozygote (rs377051298 for c.466C>T; p.Arg156Trp) missense variant were identified in the SLC4A1 and SPTA1 genes, respectively. The compound heterozygous mutations manifested as idRTA and severe HS in patient HS03. Conclusion: Our observations, for the first time, revealed clinical and genetic characteristics of idRTA and severe HS in an Iranian patient HS03.

Identification of new mutations in patients with hereditary spherocytosis by next-generation sequencing.

Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, hemolysis, splenomegaly, jaundice, and gallstones. To date, mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) have been found to be associated with different subtypes of HS. Here, we aim to investigate the presence of novel as well as known mutations in 35 Chinese patients with clinically suspected HS. Whole-exome sequencing (WES) has identified 3 patients with SLC4A1, 16 patients with ANK1, and 16 patients with SPTB mutations, including 5 splicing, 12 nonsense, 9 frameshift, 7 missense, and 1 start-loss mutation, indicating that SPTB and ANK1 are the most frequently mutated genes in Chinese HS patients. Among 34 mutations identified, 21 were novel. Most of SPTB and ANK1 mutations were nonsense (8/16) and frameshift (6/16) mutations. By trio analysis of eight families we have confirmed six de novo mutations. In addition, genotype-phenotype analysis was also performed by comparing clinical manifestations among three groups of patients with SPTB, ANK1, and SLC4A1 mutations. It revealed that patients with ANK1 mutations had a significantly higher level of MCV and MCH but lower percentage of spherocytes compared with those carrying SPTB mutations. In conclusion, our results suggested that molecular diagnosis by next-generation sequencing (NGS) is a fast, economic, and accurate way to detect and identify pathogenic alterations of inherited diseases, highlighting the potential usage of NGS in clinical practice.

[Flow cytometric test using eosin-5'-maleimide (EMA) labelling of red blood for diagnosis of hereditary spherocytosis].

OBJECTIVE: To investigate the sensitivity and specificity of eosin-5'-maleimide (EMA)assay for the diagnosis of hereditary spherocytosis (HS), and to verify the stability of reagent and samples. METHODS: EMA flow cytometry test, NaCl-osmotic fragility test and acidified glycerol lysis test were performed using peripheral blood samples from 80 patients with HS and 44 patients with other blood diseases, the sensitivity and specificity of the three methods were compared, and the feasibility of EMA binding test was estimated. The stability of EMA reagent and HS samples stored at different temperatures were tested. RESULTS: Among the 124 tested samples, the sensitivity and specificity of EMA binding test was 0.925 and 0.954, that of NaCl-osmotic fragility test was 0.950 and 0.455, and that of acidified glycerol lysis test was 1.000 and 0.318, respectively. Although the sensitivity of NaCl-osmotic fragility test and acidified glycerol lysis test was a little higher than that of EMA binding test, the specificity of the former two methods was poor, they couldn't clearly distinguish whether spherocytosis is hereditary spherocytosis. The experiment results showed that EMA was sensitive to the temperature and should not be stored in a small aliquots at -80 ℃ over a period of 6 months. The stability of the HS sample was better, 6 days storage at 4 ℃ and 3 days storage at room temperature had no influence on the results. CONCLUSION: EMA binding test by flow cytometry showed good sensitivity and specificity for HS diagnosis. EMA reagent should be stored at-80 ℃ and the HS samples should be tested within 6 days storage at 4 ℃ and 3 days at room temperature.

Novel α-spectrin mutation in trans with α-spectrin causing severe neonatal jaundice from hereditary spherocytosis.

We evaluated a neonate with severe jaundice but a negative family history. Spherocytes were present and suspected hereditary spherocytosis was confirmed by osmotic fragility and eosin-5-maleimide erythrocyte staining. We found he was a compound heterozygote for two pathogenic mutations in the gene encoding α-spectrin: a previously reported α(LEPRA) inherited from his asymptomatic mother, and a novel α-spectrin mutation in intron 45 +1 disrupting the consensus splice site, from his asymptomatic father.

Automated reticulocyte parameters for hereditary spherocytosis screening.

The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories.

Values of mean cell volume and mean sphered cell volume can differentiate hereditary spherocytosis and thalassemia.

OBJECTIVES: To determine whether the values of mean cell volume (MCV) and mean sphered cell volume (MSCV) can distinguish hereditary spherocytosis (HS) from thalassemia. METHODS: The MCV, MSCV, and other erythrocyte indexes were measured in totally 263 people, 57 HS patients, 109 thalassemia patients, and 107 normal control subjects. All indexes were derived from measurements obtained by the Beckman-Coulter LH 750 Hematology Analyzer. RESULTS: The MSCV was significantly lower in the HS group compared with the thalassemia group (P < 0.001), but the MCV was significantly higher in the HS group compared with the thalassemia group (P < 0.001). Among 57 patients with HS, the MCV was higher than the MSCV in 56 patients. The MCV was lower than the MSCV in one patient combined with ß-thalassemia. In the control and thalassemia groups, the MCV was lower than the MSCV. CONCLUSION: Measurements of the MCV higher than the MSCV can be considered an ideal index to distinguish rapidly HS from thalassemia.

Flow cytometric osmotic fragility testing does reflect the clinical severity of hereditary spherocytosis.

BACKGROUND: Osmotic fragility (OF) testing based on flow cytometry (FCM) was recently introduced for the screening of hereditary spherocytosis (HS). This study was undertaken to compare the test efficiencies of the FCM OF test and the conventional OF test, and to investigate the correlation between FCM OF results and the clinical severity of HS. METHODS: The test efficiency of FCM OF test was retrospectively compared with one of conventional OF test. FCM OF test results are expressed in two ways, that is, as percentages of residual red cells (%RRC) in hypotonic saline (%RRC values) and by expressing as a healthy individual versus patient ratio of %RRC (%RRC ratio). Cutoff values were defined using 47 subjects including 22 HS patients. HS severity scores were determined using a scoring system devised to quantify the clinical severity of HS. RESULTS: Using cutoff values of 1.68 for %RRC ratio and 61.9 for %RRC value, the test efficiency of %RRC ratio (93.6%) was higher than that of %RRC value (89.4%). However, their difference was not significant (P = 0.357). The FCM OF test (93.6%) achieved a higher test efficiency than the conventional OF test (68.9%) in 115 subjects, which included 75 HS patients (P = 0.002). %RRC ratios and %RRC values were significantly correlated with HS severity scores (P < 0.001 and P = 0.004, respectively). CONCLUSIONS: The FCM OF test was found to have greater test efficiency than the conventional OF test. Furthermore, FCM OF test results quantitatively reflected the clinical severity of HS. Using %RRC ratio is recommended to minimize false positivity although its superiority over %RRC value could not be verified statistically. The FCM OF test could be the method of choice for routine diagnostic use to screen HS and assess its clinical severity.

Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.

Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins.

A de novo interstitial deletion of 8p11.2 including ANK1 identified in a patient with spherocytosis, psychomotor developmental delay, and distinctive facial features.

The contiguous gene syndrome involving 8p11.2 is recognized as a combined phenotype of both Kallmann syndrome and hereditary spherocytosis, because the genes responsible for these 2 clinical entities, the fibroblast growth factor receptor 1 (FGFR1) and ankyrin 1 (ANK1) genes, respectively, are located in this region within a distance of 3.2Mb. We identified a 3.7Mb deletion of 8p11.2 in a 19-month-old female patient with hereditary spherocytosis. The identified deletion included ANK1, but not FGFR1, which is consistent with the absence of any phenotype or laboratory findings of Kallmann syndrome. Compared with the previous studies, the deletion identified in this study was located on the proximal end of 8p, indicating a pure interstitial deletion of 8p11.21. This patient exhibited mild developmental delay and distinctive facial findings in addition to hereditary spherocytosis. Thus, some of the genes included in the deleted region would be related to these symptoms.

Evolution of the spectrin-based membrane skeleton.

A group of four proteins - spectrin, ankyrin, 4.1 and adducin - evolved with the metazoa. These membrane-cytoskeletal proteins cross-link actin on the cytoplasmic face of plasma membranes and link a variety of transmembrane proteins to the cytoskeleton. In this paper, the evolution of these proteins is analysed. Genomics indicate that spectrin was the first to appear, since the genome of the choanoflagellate Monosiga brevicolis contains genes for alpha, beta and betaH spectrin. This organism represents a lineage of free-living and colonial protists from which the metazoa are considered to have diverged. This indicates that spectrin emerged in evolution before the animals. Simple animals such as the placozoan Trichoplax adherens also contain recognizable precursors of 4.1, ankyrin and adducin, but these could probably not bind spectrin. Ankyrin and adducin seem to have acquired spectrin-binding activity with the appearance of tissues since they appear to have largely the same domain structure in all eumetazoa. 4.1 was adapted more recently, with the emergence of the vertebrates, to bind spectrin and promote its interaction with actin. A simple hypothesis is that spectrin was prerequisite (but not sufficient) for animal life; that spectrin interaction with ankyrin and adducin was required for evolution of major tissues; and that 4.1 acquired a spectrin-actin binding activity as animal size increased with the appearance of vertebrates. The spectrin/ankyrin/adducin/4.1 complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Do we already know how spectrin attracts ankyrin?

The interaction of ankyrin and spectrin yields the major anchor between the membrane skeleton and the lipid bilayer. It is critical for red cell deformability and stability, and it is also involved in the cellular localization of several proteins, in cell differentiation, and in neuron activity. Therefore, its nature is of great interest, and recently, several researchers have had varying degrees of success in elucidating the structural basis of ankyrin-spectrin recognition. In this short paper, we briefly summarize the data obtained and compare the resulting conclusions.

Molecular epitopes of the ankyrin-spectrin interaction.

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.

An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes.

The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a beta-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa beta-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells.

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its alpha subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (alpha' spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the alpha'/alpha spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the alpha'/alpha spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/E3 activity is an important determinant of sickle cell severity.

Hereditary spherocytosis: identification of several HS families with ankyrin and band 3 deficiency in a population of southwestern Poland.

Red blood cells of 17 patients out of seven families diagnosed with HS from the southwest of Poland were studied. In six families a deficiency of ankyrin was detected, and in one family a band 3 (anion-exchanger protein) deficiency was detected. Patients from six families with the ankyrin deficiency had a 19-51% decrease in ankyrin 2.1, while the family with the band 3 deficiency showed a 33% decrease in this protein content. All changes were statistically significant, as analysed by the Student t test (P<0.05). Analysis of haemolysis kinetics gives a reliable indication of altered osmotic properties of the spherocytic cells.

[Contents of the main erythrocyte membrane proteins in patients with primary arterial hypertension and its relationship with hereditary predisposition to cardiovascular diseases]. / Soderzhanie osnovnykh belkov membran éritrotsitov u bol'nykh pervichnoi arterial'noi gipotoniei i ego sviaz' s nasledstvennoi predraspolozhennost'iu k serdechno-sosudistoi patologii.

AIM: To study quantitative content of main erythrocyte membrane proteins in patients with primary arterial hypotension and its relationship with hereditary predisposition to cardiovascular disease. MATERIAL AND METHODS: Quantitative content of main erythrocyte membrane proteins in 109 patients with primary arterial hypotension (PAH) and 124 healthy persons was measured with unidimensional polyacrylamide gel electrophoresis. Hereditary determination of PAH was studied by the clinical-genealogical method. RESULTS: PAH patients showed quantitative alterations in erythrocyte membrane protein composition: increased content of alpha-spectrin, ankyrin (band 2.1), anion exchange protein (band 3) and decreased content of actin, tropomyosin and glutathione-S-transferase. Patients with aggravated heredity for PAH had higher content of beta-spectrin and ankyrin (band 2.1 and 2.2) then patients without aggravated heredity for PAH. CONCLUSION: Quantitative alterations of membrane proteins in patients with PAH could significantly modify the structure of cytoskeleton and result in modification of the enzyme activity of transmembrane proteins (ATPases) regulating cation transport across erythrocyte membrane. Moreover, aggravated heredity for PAH predisposes to high content of cytoskeletal proteins (beta-spectrin, 2.1 and 2.2 ankyrin) which could form more compact structure of erythrocyte membrane and limit cation influx into cytoplasma.

Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi.

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

Hereditary spherocytosis with spectrin deficiency related to null mutations of the beta-spectrin gene.

Spectrin deficiency is the most common deficiency found in HS. It is heterogeneous in terms of clinical expression, inheritance (dominant or recessive) and underlying genetic defects (related to alpha- or beta-spectrin gene defects or secondary to ankyrin gene defects). We studied a sampling of French dominant HS families, selected after linkage analyses, and found the presence of mutations resulting in the silencing of the mutant beta-spectrin allele. In three HS families, one haploid set of beta-spectrin mRNA was undectectable. In two families, a deletion of 8 bases (leading to a frameshift and a premature stop codon) and a nonsense mutation were identified, respectively. In the third HS family, we were unable to characterize a relevant mutation but the loss of heterozygosity at the cDNA level suggested the presence of a null mutation of the beta-spectrin allele. Sequencing of the beta-spectrin gene has also uncovered several new polymorphisms in the coding region of the beta-spectrin gene which will be very useful for detecting loss of heterozygosity at the cDNA level and designating the beta-spectrin gene as the culprit one.

Regulation of band 3 rotational mobility by ankyrin in intact human red cells.

Ankyrin mutations and combined spectrin and ankyrin deficiency are prominent features of red blood cells (RBCs) in patients with hereditary spherocytosis (HS). Band 3 is the most abundant integral protein in the human RBC membrane. Previous studies have shown that the lateral mobility, but not the rotational mobility, of band 3 is increased in RBCs from patients with severe autosomal recessive HS and selective spectrin deficiency. These observations are consistent with the steric hindrance model of lateral mobility restriction. Here we use the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3 in intact RBCs from six patients with HS, ankyrin mutations, and combined spectrin and ankyrin deficiency. As predicted by the steric hindrance model, the lateral diffusion rate of band 3 is greater in spectrin- and ankyrin-deficient RBCs than in control cells, and the magnitude of the increase correlates with the degree of spectrin deficiency. Unlike RBCs from patients with HS and selective spectrin deficiency, however, HS RBCs with ankyrin mutations exhibit a marked increase in band 3 rotational diffusion. The magnitude of the increase correlates inversely with the ankyrin/band 3 ratio and with the fraction of band 3 retained in the membrane skeleton following detergent extraction. These data suggest that ankyrin deficiency relaxes rotational constraints on the major (slowly rotating) population of band 3 molecules. Increases in band 3 rotation could be due to release of band 3 from low-affinity binding sites on ankyrin.