Hereditary Spherocytosis Patients Research Articles

The Pyruvate Kinase Activator Mitapivat Ameliorates Anemia and Prevents Iron Overload in a Mouse Model of Hereditary Spherocytosis

Hereditary spherocytosis (HS) is the most common cause of inherited red cell membranopathy, due to mutations in genes encoding for membrane or cytoskeletal proteins, including band 3, ankyrin, spectrin, band 4.1 or band 4.2. Membrane instability results in membrane surface area loss and generation of spherocytic red cells with elevated MCHC, decreased cellular deformability and reduced red cell survival, due to splenic sequestration. Clinical management of the hemolytic anemia due to HS depends on the age of the patient and the severity of anemia. Splenectomy is indicated in children with symptomatic anemia. A classic diagnostic test for HS is the incubated osmotic fragility: this test highlights the crucial role that ATP content plays in maintenance of normal RBC function including membrane stability: it is generally believed that the increased fragility of HS is the result of abnormal ATP depletion over the 24hr incubation. We explored the hypothesis that pyruvate kinase activator, mitapivat, by modulating ATP content could have potential beneficial effects for HS RBCs in band 4.2-/- mice, a well-established model of HS (Peters LL et al JCI 103: 1527, 1999). 4.2-/- mice exhibit moderate anemia which recapitulates most of the features of typical human HS without showing the profound anemization seen in other mouse models. Oral AG-348 administration to band 4.2-/- mice at dosages of 200 mg/kg/day over 6 months resulted in (i) improvement of anemia with reduced reticulocyte count (Hb 11.6±0.035 g/dL, n=17 vs 13.104±0.09 g/dL, n=9; P<0.05; retics: 11.4±0.1%, n=16 vs 7.6±0.2%, n=7 % P<0.05) and decrease hemolytic indices (LDH, total bilirubin) with decreased spleen weight/mouse weight ratio (8.41±0.08 vs 6.53±0.06, n=8 P<0.05); (ii) reduced hepatic and splenic iron overload; (iii) reduction in the proportion of phosphatidylserine positive RBCs, measured with Annexin V binding (2.4±0.02 vs 1.5±0.06 % P<0.05); (iv) reduction of naturally occurring antibody (NAb) bound to band 4.2-/- RBC membrane. The decreased hemolysis was associated with reduction in serum erythropoietin (EPO: 1001.2±41.7 U/L vs 472 ± 12.5 U/L, n=7, P<0.05), supporting the beneficial effect of mitapivat on HS anemia. These data indicate that mitapivat ameliorates anemia of band 4.2-/- mice, reduces chronic hemolysis and improves band 4.2-/- mouse RBC features. Thus, mitapivat might represent an interesting therapeutic option for HS patients. Disclosures Kung: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Brugnara:Sysmex America Inc.: Consultancy; American Journal of Hematology: Other.

Genetic and Clinical Characteristics of Patients With Hereditary Spherocytosis in Hubei Province of China.

Hereditary spherocytosis (HS) is an inherited disorder characterized by anemia, splenomegaly, and spherical-shaped erythrocytes, caused by mutations in erythrocyte membrane Protein Genes (ANK1, SPTB, SLC4A1, SPTA1, and EPB42). We investigated molecular spectrum and genotype-phenotype correlation in HS patients in Hubei province, central China. Twenty-three patients with HS were included. A next-generation sequencing (NGS) panel targeting ANK1, SPTB, SLC4A1, SPTA1, and EPB42 genes was used to screen potential variants. Sanger sequencing was applied to validate variants. Of the twenty-three patients, thirteen patients carried ANK1 variants, and ten patients harbored SPTB variants, including ten non-sense, six indel, four splice site, one start-loss, and one missense variant. Four out of twenty-two variants in our study were known, and eighteen variants were novel. Most ANK1 and SPTB variants were indel (5/12) or non-sense (7/10), respectively. Family member analysis in thirteen families showed that six variants were de novo. Variable expressivities were observed in a pair of twins with ANK1 c.341C > T variant, and two unrelated patients both carried ANK1 c.2T > A variant. Genotype-phenotype analysis found no significant difference between ANK1 and SPTB regarding the levels of Hb, RBC, MCV, MCH, and MCHC. However, variants in the ANK1 death domain were associated with lower levels of MCV and MCH compared to other ANK1 domains. In conclusion, NGS is a fast way to provide a molecular HS diagnosis. We also identified unique genetic and clinical characteristics of patients with HS in Hubei Province, China. However, a large sample size is needed to further investigate the genotype-phenotype correlation.

Usefulness of Eosin-5’-Maleimide (EMA) Dye for the Diagnosis of Hereditary Spherocytosis

Abstract Background the normal biconcave discoid shape of the red blood cell (RBC) is maintained by cytoskeletal proteins underneath the membrane. This protein network and its link with the lipid bilayer are essential for RBCs to remain flexible and carry out their physiological functions. A number of disorders including hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) are caused by decreased expression or mutation of cytoskeletal proteins. Aim of the Work to evaluate the usefulness eosin-5-maleamide (EMA) dye binding flow cytometric test in the diagnosis of HS and assess the sensitivity and specificity of this test to distinguish HS patients from patients having anemia due to other causes. Subjects and Methods this study was conducted on 52 patients with chronic anemia, 21 diagnosed as HS (group I) and 31 patients with anemia due to other causes (group II). The patients were 28 males and 23 females with a male to female ratio 1.2:1, their ages ranged between 3 to 12.5 years old attending at the Pediatric Hematology Clinics, Ain Shams University Hospitals. Normal control group (group III) was included in the study. Results median fluorescence intensity (MFI) had shown significantly lower values in HS patients (group I) (3.78±0.48) than (iron deficiency anemia) IDA patients (group IIa) (5.56±0.35), auto immune hemolytic anemia (AIHA) patients (group IIb) (5.7±0.27), beta thalassemia intermedia (BTI) patients (group IIc) (6.01±0.63) and control group (group III) (5.3±0.43). The % reduction in EMA fluorescence proved to be significantly higher in HS patient (group I) (29.88±8.27) than in IDA patients (group IIa) (1.13±6.18), AIHA patients (-6.14±10.44) and BTI patients (group IIc) (-5.1±10.07). Multi regression analysis had been used to check the most sensitive predictors for diagnosis of HS, both EMA MFI and the % reduction in EMA fluorescence proved to be the best sensitive predictive values for discriminating HS patients, however the higher F-ratio in EMA MFI (F = 25.222) than in % reduction in EMA fluorescence intensity (F = 19.866) showed that the MFI is even better than % reduction in fluorescence in diagnosis of HS. Conclusion the present study highlighted the role of flow cytometric EMA binding test in differentiating HS patients from patients having anemia due to other causes.

Splenic function is not maintained long-term after partial splenectomy in children with sickle cell disease

BackgroundPartial splenectomy (PS) may allow preservation of splenic function in cases where splenectomy is indicated for hematologic diseases; however, the long-term outcomes are uncertain. We investigated the long-term outcomes of PS in patients with sickle cell disease (SCD). MethodsA single-institution retrospective chart review was performed for children with SCD who underwent PS from 1997 to 2017. For comparison, we reviewed outcomes for patients who underwent PS for hereditary spherocytosis (HS). The primary endpoint was viability of the splenic remnant as inferred by the presence of remnant perfusion on ultrasound and/or liver spleen scan. ResultsNine patients with SCD and 26 patients with HS underwent PS at a median age of 11 (IQR, 9–14) and 7.5 (IQR, 6–13) years, respectively. All underwent laparoscopic PS with three (7.9%) conversions to open. Two SCD patients were lost to long-term follow-up. The remaining seven SCD patients had initial postoperative splenic remnant perfusion demonstrated by ultrasonography. By 42 months postoperatively, however, none had a functioning splenic remnant. The median time to loss of splenic remnant was 12.6 (IQR 9.2–28.5) months. In contrast, all HS patients demonstrated robust splenic remnant blood flow with a median follow-up of 46 (IQR 37–82) months. ConclusionNo patient with SCD who underwent PS had viable splenic tissue for more than 42 months, likely due to continued autoinfarction typical of patients with this disease. Therefore, we believe that PS to preserve splenic function is not indicated in patients with SCD. Level of evidenceIII.

Linkage of typically cytosolic peroxidases to erythrocyte membrane – A possible mechanism of protection in Hereditary Spherocytosis

Hereditary Spherocytosis (HS) is a non-immune hemolytic anemia associated to oxidative stress (OS), namely to the linkage of cytosolic antioxidant enzymes to the erythrocyte membrane. Our aims were to evaluate erythrocyte OS changes and the membrane linkage of peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) in unsplenectomized (unspl) and splenectomized (spl) HS patients and to search for associations with clinical severity (in unspl HS patients). We studied 114 HS patients (74 unspl and 40 spl) and 30 healthy individuals and we evaluated membrane bound hemoglobin (MBH), membrane lipid-peroxidation (LPO), enzymatic activities of GPx and CAT and the amounts of membrane bound Prx2, GPx and CAT, as well as, clinical and analytical parameters for characterization of HS. We found that unspl HS patients showed clear signs of anemia and in spl HS, a correction to this anemia was observed; the latter patients presented higher levels of OS biomarkers, namely, MBH and LPO. CAT was detected in the membrane of all individuals (control and HS groups), while GPx and Prx2 were only present in HS patients; moreover, their linkage to the membrane (in HS) appears to be cumulative since membrane bound peroxidases amount was higher as the number of peroxidases detected increased. MBH increased with the number/amount of membrane bound peroxidases, however LPO levels remained similar. In conclusion, our data suggest that the binding of these typically cytosolic peroxidases to erythrocyte membrane may be part of a mechanism of membrane protection to maintain its integrity by possibly regulating LPO.

The Complexity of Genotype-Phenotype Correlations in Hereditary Spherocytosis: A Cohort of 95 Patients: Genotype-Phenotype Correlation in Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is a phenotypically and genetically heterogeneous disease. With the increased use of Next Generation Sequencing (NGS) techniques in the diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. In this study, we aimed to explore genotype-phenotype correlation in 95 HS patients genotyped by targeted NGS as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands). In 85/95 (89%) of patients a pathogenic mutation was identified, including 56 novel mutations. SPTA1 mutations were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 (α-spectrin) mutations showed autosomal dominant inheritance. ANK1 (ankyrin1) mutations accounted for 27% (23/85 patients) and SPTB (β-spectrin) mutations for 20% (17/85 patients). Moderate or severe HS was more frequent in patients with SPTB or ANK1 mutations, reflected by lower hemoglobin concentrations and higher reticulocyte counts. Interestingly, mutations affecting spectrin association domains of ANK1, SPTA1 and SPTB resulted in more severe phenotypes. Additionally, we observed a clear association between phenotype and aspects of red cell deformability as determined by the Laser assisted Optical Rotational Cell Analyzer (LoRRca MaxSis). Both maximal deformability and area under the curve were negatively associated with disease severity (respectively r = -0.46, p < 0.01, and r = -0.39, p = 0.01). Genotype-phenotype prediction in HS facilitates insight in consequences of pathogenic mutations for the assembly and dynamic interactions of the red cell cytoskeleton. In addition, we show that measurements of red blood cell deformability are clearly correlated with HS severity.

PS1200 IDENTIFICATION OF MUTATIONS IN RED CELL MEMBRANOPATHIES USING NGS‐TARGETED SEQUENCING PANEL

Background:Red cell membranopathies refers to genetically and phenotypically heterogenous disorders due to defects in red cell membrane and skeletal proteins. Phenotypes vary from severe transfusion‐dependent hemolytic anemia to fully compensated hemolysis. Causative genes implicated in RBC membranopathies are numerous, making a gene‐by‐gene approach time consuming, expensive and labor intensive. Use of Next Generation Sequencing (NGS) targeted sequencing panel can expedite the molecular diagnosis after routine laboratory tests.Aims:This study aimed at defining a strategy for effective diagnosis of RBC membrane defects and identification of pathogenic mutations associated with the disease.Methods:Patients with clinical symptoms of hemolytic anemia, jaundice, hepatosplenomegaly and some requiring frequent blood transfusions were recruited in this study. Blood was collected from the patients after informed consent. Peripheral blood smear examination and flow cytometric based EMA binding test were performed and identification of mutations in red cell membrane proteins was done using NGS‐targeted sequencing panel. The relationship between gene mutations and clinical phenotypes was analyzed by In silico prediction tools such as SIFT, Polyphen2.Results:We identified mutations in 21 cases of Hemolytic Anemia (HA) in membrane protein‐coding genes using NGS‐targeted sequencing panel. Five Hereditary Spherocytosis (HS) patients were diagnosed with mutations in ankyrin (ANK1) gene, 3 patients with alpha spectrin (SPTA1) gene and 4 patients with beta spectrin (SPTB) gene. Band 3 (SLC4A1) gene mutations was identified in two HS cases and in 2 with dRTA cases. Five patients showed mutation in PIEZO1 gene causing Hereditary xerocytosis (HX). Twelve novel mutations were found as shown in Table 1.Summary/Conclusion:RBC membranopathies showed highly varied etiology. Our experience demonstrates the high diagnostic yield of NGS for molecular diagnosis of RBC membrane disorders associated with defects in large genes. Timely detection of the disorder not only offers therapeutic benefits for our patients but is likely to help in genetic counseling and future antenatal diagnosis, if required.image

S902 THE COMPLEXITY OF GENOTYPE‐PHENOTYPE CORRELATIONS IN HEREDITARY SPHEROCYTOSIS: A COHORT OF 95 PATIENTS

Background:Hereditary spherocytosis (HS) presents as a highly heterogeneous disease, both phenotypically and genetically. Disease severity is directly related to the extent of loss of surface area of the red cell. The diagnosis is multifaceted and includes functional testing of red cell deformability. Until recently, establishing or confirming the diagnosis at the DNA level was only rarely done. With increased use of Next‐Generation‐Sequencing (NGS) techniques in diagnosis of red blood cell disorders, the list of unique pathogenic mutations underlying HS is growing rapidly. However, insight into the impact of genetic defects on the function of membrane proteins, and consequently on phenotypic presentation, is still limited.Aims:Explore genotype‐phenotype correlation in a large cohort of patients with HS.Methods:Retrospective analysis on a cohort of 95 HS patients genotyped by targeted‐NGS (SPTA1, SPTB, ANK1, SLC4A1, EPB41, EPB42, RHAG) as part of routine diagnostics (UMC Utrecht, Utrecht, The Netherlands).Results:Our cohort included 95 patients suspected to have HS. In 85/95 (89%) of the patients a pathogenic mutation was identified, including 56 novel mutations. Mutations in SPTA1 were most frequently encountered (36%, 31/85 patients), primarily in patients with autosomal recessive forms of HS. Three SPTA1 mutations showed autosomal dominant inheritance. ANK1 mutations accounted for 27% (23/85 patients) and SPTB mutations for 20% (17/85 patients) of the cases, all showing autosomal dominant inheritance. Moderate or severe HS was more frequently observed in patients with SPTB or ANK1 mutations, reflected by significantly lower hemoglobin concentrations (SPTB 10.5 g/dL, ANK1 10.8 g/dL compared to SLC4A1 13.2 g/dL, both p &lt; 0.05) and higher absolute reticulocyte counts (SPTB 580x10^9/L, ANK1 506x10^9/L compared to SPTA1 298x10^9/L and SLC4A1 259x10^9/L, all p &lt; 0.01) in non‐splenectomized patients.We investigated the correlation between phenotype and genetic mutations using protein structure information (Figure). Mutations positioned in the spectrin‐association domains of ANK1, SPTA1 and SPTB resulted in a more severe phenotype. Interestingly, deletion of the whole SPTB gene resulted in a mild phenotype, while the majority of other pathogenic mutations in SPTB were associated with a more severe disease phenotype, suggesting that incorporation of truncated or dysfunctional protein occurs, which might be more harmful than an absolute decrease of otherwise normal protein.Our cohort included nine families and broad phenotypic heterogeneity was observed in four of them. Phenotypic heterogeneity could not be directly explained by the underlying genotype, suggesting that other (genetic) yet‐unknown modifiers contribute to disrupted interactions in the red cell cytoskeleton. To identify alternative predictors of clinical severity we analyzed red cell Osmoscan profiles from the Laser‐assisted Optical Rotational Cell Analyzer. Data were available in a subset of our cohort (44 not splenectomized patients). We observed a clear association between clinical disease severity and the maximal deformability, and the area under the curve (respectively r = −0.48, p &lt; 0.01, and r = −0.38, p = 0.01).Summary/Conclusion:Genotype‐phenotype prediction in HS requires additional insight in the consequences of pathogenic mutations for assembly of and dynamic interactions in the red blood cell cytoskeleton. Currently, in conjunction to the molecular deficit, functional analysis of red cell deformability adds to the prediction of phenotypic variability.image

PS1196 INNOVATIVE DIAGNOSTIC APPROACH FOR RARE ANEMIA

Background:Rare anemia (RA) embrace a highly heterogeneous group of ultra‐rare disorders which severe forms are life‐threatening with complex genotype‐phenotype correlations. Both conventional and NGS present bottlenecks.Aims:To establish an integrative diagnostic algorithm encompassing conventional and new methodologies for reducing the number of misdiagnosed/undiagnosed cases.Methods:53 patients with chronic hemolytic/hyporegenerative anemia included and categorized based on a) clinical criteria b) EMA‐Binding test (EBT) and osmotic gradient ektacytometry (LoRRca) and c) t‐NGS (69 disease causing and candidate genes).EBT conventional cut‐off for Hereditary Spherocytosis (HS) was used (decreased fluorescence &gt;21%). LoRRca cut‐offs already established by the group. Genetic variants (coverage &gt;100x) were annotated on variant effect and MAF and IGV assessed. Disease causing variants on inheritance pattern and VarSome plus Human Splicing Finder. Interpretation and nomenclature according to ACMG. t‐NGS was considered as gold standard for conclusive diagnosis and tests sensitivity.Results:Clinical orientation/conclusive diagnosis correlation is shown in the figure.HS oriented patients were stratified according to CPG: 1 to 4 HS diagnosis criteria (family history, peripheral spherocytes, raised MCHC, % hyperdense). Only 5/43 oriented HS patients fulfill all criteria, in which EBT, LoRRca and t‐NGS confirmed HS diagnosis. EBT was HS positive in 31 of 38 remaining cases (81.6%) while LoRRca showed HS pattern in the 38. After t‐NGS, conclusive HS diagnosis was reached in 36 of the 38 cases. 5 of them also showed a disease causing mutation on RBC enzymes. 2 cases remained undiagnosed, both with EBT and LoRRca HS compatible profiles. In addition, correlation among EBT and HS causative genes showed lower range of values (x = −19%) for SPTA1 variants in contrast with SLC4A1 variants (x = −35%). Five from 6 clinically oriented as Hereditary Elliptocytosis (HE) were confirmed by t‐NGS, resulting in 3 HE and 2 sever forms, Hereditary Piropoikilocytosis (HPP). EMA BT was HS positive in one HPP case showing a double population. LoRRca showed HE profile in 5 cases and HS profile in 1 case corresponding with the third undiagnosed case which showed a disease causing CDAN1 variant and a SPTB VUS in heterozygous status. One case oriented ad Hereditary Xerocytosis (HX) was diagnosed by both, LoRRca and t‐NGS, with a mild form of HX. From the two cases oriented as chronic non‐spherocytic hemolytic anemia, 1 was diagnosed by t‐NGS as HX (LoRRca and EBT negative) and 1 correspond with the fourth undiagnosed case which presented EBT in the “gray zone” and non‐conclusive LoRRca profile. In 1 patient with compensated hemolysis, conclusive diagnosis was reached based on a HBA1 variant coding for an unstable Hb (Evans) causing mild hemolytic anemia. Genetic diagnosis of 92% (49/53) of the included cases could be determined. The 4 undiagnosed patients will be further analyzed by 33 candidate genes and WES (ERN‐EuroBloodNet –SOLVE). LoRRca showed a sensitivity of 100% for HS (83% for HE). EBT showed a total sensitivity of 85% for HS (cut‐off &gt;21%) while increasing the cut‐off to &gt;16% would increase sensitivity to 93% without increasing false positive, confirming results already reported (Llaudet, 2017).Summary/Conclusion:Establishing an integrative harmonized diagnostic algorithm, including LoRRca and t‐NGS has been revealed essential for high diagnosis performance and reduce misdiagnosis, thus, directly impacting on better personalized diagnosis for RA.image

PS1204 SEVERE HEREDITARY HAEMOLYTIC ANAEMIA DUE TO THREE NOVEL SPTA1 MUTATIONS IN TWO COMPOUND HETEROZYGOUS UNRELATED PATIENTS

Background:Red blood cell (RBC) membrane defects are a cause of hereditary haemolytic anaemia (HHA) due to mutations in the genes that encode cytoskeletal proteins leading to a reduced cell deformity. Using next generation (NGS) and whole exome sequencing (WES), in two unrelated patients with HHA due to hereditary pyropoikilocytosis (HPP) and hereditary spherocytosis (HS), we have identified a complex set of mutations that affect SPTA1. Three are new: two due to an abnormal splicing and one to a micro election of chromosome 1Aims:The main objective of our task is to provide a fast and efficient diagnosis of HHA using NGS and WESMethods:NGS that includes a panel of 35 genes responsible for membranophaty (ANK1, EPB41, EPB42, SLC4A1, SPTB, SPTA1), haemoglobinopathy (HBA1, HBA2, HBB), enzymopathy (ADA, AK1, ALDOA, BPBM, CYB55, G6PD, GCLC, GPI, GSR, GSS, HK1, NT5C3A, PFKM, PGK1,) and CDA (CDAN1, C15orf41, SEC23B, KLF1, GATA1, KIF23). NGS and WES have been performed using an Illumina HiSeq2000 deviceResults:Patient 1. A 40 year‐old woman with HPP. CBC: Hb: 102 g/l, MCV: 101fl and Retics: 260 x109/l. The study was performed after splenectomy and the OGE curve was typical of severe HS. NGS revealed two canonical splice‐site mutations in SPTA1 gene: c.6842+2T&gt;C and c.1599+2T&gt;C. The c.6842+2T&gt;C mutation is found in the lateral nucleation zone of a‐spectrin with b‐spectrin and produces an aberrant splicing eventually in 100% of the cases with a production of α‐chains unsuitable for spectrin dimerization, and a decrease of binding to skeleton during the heterodimers assembly. The c.1599+2T&gt;C mutation is in the zone of association of spectrin dimers leading to an aberrant splicing eventually in 100% of the cases and probably a change in the protein conformation that produces a breakage of tetramerisation and a loss of skeleton stabilityPatient 2. A 2 year‐old boy with HS. CBC: 72 g/l, MCV 95 fl and Retics:182 x109/l. Splenomegaly. The OGE curve was typical of moderate HS. The NGS analysis demonstrated a coinheritance for two different mutations in SPTA1 gene: n.158537345_158849273del and c.4339–99C&gt;T. The n.158537345_158849273del, confirmed by WES, leads to a micro deletion of chromosome 1 that removes the entire SPTA1, leading to the absence of α‐spectrin. The c.4339–99C&gt;T, also known as spectrin α‐LEPRA, is an autosomic recessive mutation that causes a decrease of a‐chain of 84%. Both SPTA1 gene mutations are autosomal recessive and their co‐inheritance explains the severe HS.Summary/Conclusion:HPP and H are heterogeneous disorders of RBC membrane that can result from mutations in the genes encoding α‐spectrin (SPTA1). The resulting defects an abnormal horizontal and/or vertical cytoskeletal associations affecting the stability and deformability of RBC membrane. The clinical diagnosis of HE and HS is based on the identification of the characteristic RBC morphology and specific membrane rheological abnormalities using the OGE. Here, two unrelated patients with severe elliptocytosis (HPP) and HS are described with the joint inheritance of a complex set of mutations (four in total) in the SPTA1 gene; three are new and one is the known as α‐LEPRA. The correlation between genetic mutations NGS and EOG is discussed. Our study demonstrates the existence of complex interactions between SPTA1 gene mutations leading to severe haemolytic anaemia highlighting the usefulness of molecular diagnostics in patients with no identifiable family history of disorders of the erythrocyte cytoskeleton

PF507 THE HEPCIDIN‐ERYTHROFERRONE AXIS AND ITS RELATIONSHIP WITH ERYTHROPOIESIS IN DIFFERENT TYPES OF ANEMIA

Background:The regulation of iron stores depends on hepcidin, while the adaptation mechanisms of iron availability in case of anemia are mediated by an erythroid factor that could be erythroferrone (ERFE). ERFE is produced by erythroid precursors in the bone marrow and the spleen in response to erythropoietin (EPO) stimulation and bleeding in animal models. ERFE induces hepcidin suppression during increased erythropoietic activity and thereby increases iron availability for new erythrocytes synthesis. Therefore, a negative correlation between serum ERFE concentrations and Hb level and serum ferritin were observed in pediatric patients with iron deficiency anemia (IDA). The role of so‐called hepcidin‐ERFE axis have not been confirmed in cases of increased and sustained erythropoiesis, like congenital hemolytic or dyserythropoietic anemias.Aims:To evaluate hepcidin and ERFE levels in different erythroid disorders to investigate their possible implications on iron metabolism.Methods:Multicentric study, in which 5 groups of patients were included: 1) healthy controls, 2) IDA, 3) congenital hemolytic anemias with biochemical data of active hemolysis, 4) congenital anemias with ineffective erythropoiesis (non‐transfusion dependent thalassemia, dyserythropoietic anemia) and 5) hereditary spherocytosis (HS) previously treated with splenectomy. In all groups, determination of hemoglobin, ferritin, hepcidin and ERFE levels was performed. The quantitative measurement of hepcidin in serum was a solid phase enzyme‐linked immunosorbent assay based on the principle of competitive binding, with a detection range 0.153–81 ng/ml and sensitivity 0.153 ng/ml. The quantitative measurement of ERFE (FAM132B) in serum was based on a standard sandwich enzyme‐linked immunosorbent assay technology with a detection range 156–10,000 pg/ml and sensitivity 75 pg/ml.Results:A total of 94 patients were included, of which 33 were excluded (distributed in all groups) because they did not have a valid ERFE value (not detectable with the method used). The 61 remaining cases distributed as follows: Healthy controls 26 (42.6%), IDA 9 (14.8%), congenital hemolytic anemia 13 (21.3%), ineffective erythropoiesis 4 (6.5%), splenectomized HS patients 9 (14.8%). The median values of Hb, ferritin, hepcidin and ERFE of the different groups are summarized in table 1.Summary/Conclusion:In IDA patients, a statistically significant decrease in hepcidin is observed; otherwise, there is an increase in ERFE, although not statistically significant, that could be explained by the low number of samples analyzed. In hemolytic anemias, despite an increase in erythropoiesis, there is no increase in ERFE, which may be in relation to the absence of anemia (compensated hemolytic anemia) and the increase of hepcidine could be explained by the high ferritin levels. In the ineffective erythropoiesis group, despite the anemia, no statistically significant ERFE‐hepcidin levels were observed. In splenectomized HS patients, ERFE/hepcidine levels were similar to those of the control group.imageThe newly identified ERFE hormone may act as physiological hepcidin suppressor in cases with IDA, but we have to establish this relationship in other types of anemia, for which it is necessary to evaluate more patients. The high number of patients that we had to exclude due to ERFE results outside the assay sensitivity range implies the need of validation and standardization of the method.

Hereditary spherocytosis and allied disorders

Hereditary spherocytosis and allied disorders

PS1199 SCREENING FOR HEREDITARY SPHEROCYTOSIS IN DAILY PRACTICE: WHICH IS THE BEST ALGORITHM USING ERYTHROCYTE AND RETICULOCYTE PARAMETERS?

Background:Hereditary spherocytosis (HS) is the most common inherited chronic haemolytic anemia in Northern Europe. Its prevalence is estimated to 1 in every 2000 births. The actual guidelines for the screening and diagnosis of HS are based on clinical and family histories, physical examination and laboratory findings. Additional parameters based on complete blood count (CBC) and reticulocyte parameters have been developed on haematology analyzers leading to many publications about their effectiveness as HS screening tool.Aims:We evaluated and compared the effectiveness of four published algorithms for the screening of HS using two haematology analyzers i.e. the UniCel DxH800 (DxH; Beckman‐Coulter, Miami, FL) and the XN‐9000 (XN; Sysmex, Kobe, Japan) and determined which algorithm could be the most suitable in our daily clinical practice.Methods:A total of 96 EDTA samples obtained for HS screening were analyzed prospectively on both haematology analyzers and a CBC with extended reticulocyte parameters were collected. These included 12 confirmed HS patients and 84 non‐HS patients. The diagnosis of HS was based on clinical and family histories, cryohaemolysis test, eosin‐5’‐maleimide (EMA) dye binding test, ektacytometry and identification of the defective protein. The specific reticulocyte parameters used on the DxH800 were mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV), and on the XN‐9000 were hypohaemoglobinized erythrocytes (Hypo‐He), microcytic erythrocytes (MicroR) and IRF. For each algorithm, we established sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV).Results:The results are reported in Table 1. The only algorithm that provided a sensitivity of 100% was the algorithm published by Lazarova et al. in 2014 using the DxH800. The three algorithms published based on the Sysmex parameters showed much lower performances than the one based on the DxH800 parameters i.e. out of the 96 patients with HS, between 2 to 6 patients were screened as negative for HS. Three of the published algorithms were evaluated on a previous XE model i.e. XE‐5000, mode of HS diagnosis is not always comparable as well as the amount of patients involved in the different studies.Table 1: Performance characteristics of the different algorithms (original stated performance characteristics) tested prospectively on 12 HS and 84 non‐HS patients.Summary/Conclusion:To screen for HS in the daily practice, the extended reticulocyte parameters provided by the Sysmex analyzers seem promising but the clinical cut‐offs on the XN‐9000 should be reevaluated on a larger number of patients.image

Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis.

Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or β-spectrin using red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis.

The hemostasis system in children with hereditary spherocytosis

IntroductionPatients with hereditary spherocytosis (HS) are characterized by having an increased risk for thrombosis. An early manifestation of thrombotic complications can occur even in childhood, especially after surgery. Hypercoagulability can be associated with hemolytic crises. AimThe aim of this study was to investigate the hemostatic state in children with HS using global hemostasis assays. MethodsThe hemostatic status of 62 children (38 boys and 24 girls; age range: 0.5 to 17 years) with HS during and without hemolytic crisis was assessed using clotting times (APTT, TT, and PR), fibrinogen and D-dimer levels, and global hemostasis, thromboelastography (TEG) and thrombodynamics (TD) assays. One hundred and two healthy children undergoing annual medical examination were enrolled as a control group. ResultsTEG and TD parameters were increased in the children with HS compared to the control group (60 ± 5 mm vs. 53 ± 4 mm, p < 0.05 for TEG maximum amplitude; 28 ± 3 μm/min vs. 24 ± 2 μm/min, p < 0.05 for TD clot growth rate), while APTT, TT and PR were not significantly different between the two groups. Patients with HS were divided into 2 groups: those during hemolytic crisis (28 patients) and those without hemolytic crisis (34 patients). TEG and TD parameters were increased in those during hemolytic crisis compared to the steady state HS group (62 ± 5 mm vs. 57 ± 4 mm, p < 0.05 for TEG maximum amplitude; 31 ± 4 μm/min vs. 26 ± 3 μm/min, p < 0.05 for TD clot growth rate). The D-dimer levels were increased in 4 HS patients, for whom the activation of blood clotting was noted. Fibrinogen levels were decreased in patients with HS compared to the control group (2.1 ± 0.4 mg/ml vs. 2.6 ± 0.4 mg/ml, p < 0.05). Other tests were within the reference ranges for both groups. ConclusionsThe global hemostasis tests TEG and TD revealed hypercoagulability in patients with HS. More dramatic changes were observed in patients experiencing a hemolytic crisis.

Whole exome sequencing identified a novel mutation (p.Ala1884Pro) of β-spectrin in a Chinese family with hereditary spherocytosis.

Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte. The typical feature of HS is the presence of spherical-shaped erythrocytes on the peripheral blood smear. According to previous studies, more than five candidate genes, such as ANK1, SPTB, SPTA1, SLC4A1 and EPB42 have been identified in HS patients. In the present study, a Chinese HS family was investigated. The proband suffered from pathologic jaundice and splenomegaly. A blood test and peripheral blood smear experiment further confirmed the diagnosis of HS. We selected the proband to perform the whole exome sequencing. After data filtering and co-segregation analysis, we identified 12 mutations in affected members that were absent in healthy members. In consideration of the inheritance pattern, Online Mendelian Inheritance in Man clinical phenotypes, Toppgene function and American College of Medical Genetics classification, we considered the novel mutation (c.5650G>C/p.Ala1884Pro) of β-spectrin (SPTB) to be the genetic lesion in this family. The novel mutation, resulting in a substitution of alanine by proline, may lead to transformation of the SPTB protein structure, which affects the binding between SPTB and ankyrin. The present study confirmed the hereditary red blood cell membrane disorders at a molecular level and expanded the spectrum of SPTB mutations. This may contribute to the clinical management and genetic counseling with respect to HS.

Disordered bone metabolism in hereditary spherocytosis patients

ABSTRACTAim: This study was planned to evaluate bone health in patients with hereditary spherocytosis.Materials and methods: In this prospective study, a total of 30 hereditary spherocytosis patients which followed in the Pediatric Hematology and Oncology Department of KSU Medical Faculty and 30 patients for control group were included. Patient and control group were chosen equal in age and sex. Hemogram and biochemical tests (serum calcium, phosphorus, alkaline phosphatase, parathormone, vitamin D) and osteocalcin were studied from the patient and control groups. Also DXA examination was performed in the patient group.Results: There was a significant difference in hemogram parameters between the two groups due to hemolytic anemia in hereditary spherocytosis patients. In the patient group, osteocalcin was 6.88 ± 4.35 ng/ml, vitamin D was 17.74 ± 7.76 ng/ml and in the control group osteocalcin was 11.93 ± 8.92 ng/ml, vitamin D was 24.04 ± 11.70 ng/ml. There was a statistically significant difference between the vitamin D and osteocalcin levels of the two groups (p = 0.017 and 0.008, respectively). Bone density was assessed in the patient group. In patients DXA results showed lower Z-scores then the normal population according to age and sex.Conclusion: Hereditary spherocytosis patients should be followed closely in terms of development, puberty, bone health as they are in other hemolytic anemias. Nutritional recommendations, vitamin D supplementation, physical activity should be advised to protect bone health.

Application of High Resolution Melting Curve Analysis in Detection of SLC4A1 Gene Mutation in Patients with Hereditary Spherocytosis

To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.

The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis

Extracellular vesicles (EVs) are widely studied regarding their role in cell-to-cell communication and disease, as well as for applications as biomarkers or drug delivery vehicles. EVs contain membrane and intraluminal proteins, affecting their structure and thereby likely their functioning. Here, we use atomic force microscopy for mechanical characterization of erythrocyte, or red blood cell (RBC), EVs from healthy individuals and from patients with hereditary spherocytosis (HS) due to ankyrin deficiency. While these EVs are packed with proteins, their response to indentation resembles that of fluid liposomes lacking proteins. The bending modulus of RBC EVs of healthy donors is ~15 kbT, similar to the RBC membrane. Surprisingly, whereas RBCs become more rigid in HS, patient EVs have a significantly (~40%) lower bending modulus than donor EVs. These results shed light on the mechanism and effects of EV budding and might explain the reported increase in vesiculation of RBCs in HS patients.

Clinical manifestations of erythrocyte membrane protein coding gene mutations in hereditary spherocytosis

目的探讨遗传性球形红细胞增多症（HS）红细胞膜蛋白基因突变与患者临床表现的关系。方法对25例HS患者的HS常见突变进行靶向测序，并根据突变与患者临床特征评估两者相关性。结果25例患者中男13例，女12例，中位年龄20（4~55）岁。中位HGB为105（65~150）g/L，其中代偿性溶血9例，轻度贫血9例，中度贫血3例，重度贫血4例。18例（72%）患者携带与HS致病相关的基因突变，包括6例ANK1突变、6例SLC4A1突变、5例SPTB突变和1例EPB41突变；7例（28%）无相关突变。SPTB突变与SLC4A1突变患者中以男性为主，携带基因突变的HS患者发病年龄（P=0.130）与HGB水平（P=0.585）与无突变患者差异无统计学意义，但EMA标记缺失的程度（P=0.015）、酸化甘油溶血试验（AGLT50）（P=0.032）以及红细胞渗透脆性试验（EOF）开始溶血浓度（P=0.027）显著高于无突变患者。结论HS红细胞膜蛋白基因突变筛查对明确诊断有指导意义，无突变患者临床溶血表现较轻。