Red Blood Cell Enzyme Research Articles

Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

Pyruvate kinase deficiency in children.

Pyruvate kinase deficiency (PKD) is a rare, autosomal recessive red blood cell enzyme disorder, which leads to lifelong hemolytic anemia and associated complications from the disease and its management. An international, multicenter registry enrolled 124 individuals younger than 18years old with molecularly confirmed PKD from 29 centers. Retrospective and prospective clinical data were collected. There was a wide range in the age at diagnosis from 0 to 16years. Presentation in the newborn period ranged from asymptomatic to neonatal jaundice to fulminant presentations of fetal distress, myocardial depression, and/or liver failure. Children <5years old were significantly more likely to be transfused than children >12 to <18years (53% vs. 14%, p=.0006), which correlated with the timing of splenectomy. Regular transfusions were most common in children with two severe PKLR variants. In regularly transfused children, the nadir hemoglobin goal varied considerably. Impact on quality of life was a common reason for treatment with regular blood transfusions and splenectomy. Splenectomy increased the hemoglobin and decreased transfusion burden in most children but was associated with infection or sepsis (12%) and thrombosis (1.3%) even during childhood. Complication rates were high, including iron overload (48%), perinatal complications (31%), and gallstones (20%). There is a high burden of disease in children with PKD, with wide practice variation in monitoring and treatment. Clinicians must recognize the spectrum of the manifestations of PKD for early diagnostic testing, close monitoring, and management to avoid serious complications in childhood.

Assessment of serum Zinc, Copper and Selenium in non-symptomatic Sickle-Cell Anaemia patients at the University Teaching Hospital, Lusaka, Zambia

Background: Zinc, copper and selenium are known important trace elements playing a vital role as cofactors of enzymes such as superoxide dismutase and glutathione peroxide. They form part of the first line antioxidants enzymes in red blood cells and plasma. The study aimed at determining the serum levels of zinc, copper and selenium in asymptomatic sickle-cell anaemia patients. Methods: This was a case control study conducted from the specialist Haematology and Oncology Clinic 4 at the University Teaching Hospital, Lusaka, Zambia. 5mls of whole blood was collected from 46 sickle-cell anaemia patients and 46 healthy controls from Out-Patient Department after consent. Using Atomic Absorption Spectrometry, serum levels of zinc, copper and selenium was determined. Independent t-test was used to compare the means and a binary logistic regression for the likelihood of developing a sickling crisis, using SPSS version 20.Results: The mean serum levels of zinc in patients were lower 86.92±20.46mg/L against 91.33±43.23mg/L; p&lt;0.028 of controls. Copper mean levels were 149.85±54.82mg/L against 134.32±54.16mg/L; p&lt;0.191 of controls. Selenium mean levels were 0.100±0.041mg/L against 0.081±0.032mg/L; p&lt;0.380 in patients compared to controls. The odds of developing one sickling crises in the past year given the levels of zinc, copper and selenium was [B = -.340, zinc Exp(B) =1.011, copper Exp(B) = 1.008, selenium Exp(B) = .000, DF = 1].Conclusion: Zinc is reduced in sickle-cell anaemia patients. The probability of developing a sickling crisis is lower in patients with higher serum levels of zinc and copper by 1.1% and 0.8% respectively.

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Forms of pseudotuberculosis progression in children and the impact of lipid peroxidation activation and antioxidant system imbalance on their development

The objective is to compare clinical symptoms, lipid peroxidation indicators, the state of the antioxidant system and assess their impact on the severity and progression of pseudotuberculosis in children.Materials and methods. We examined 125 children with pseudotuberculosis divided into 4 groups according to the severity and nature of the disease progression and 45 healthy children. The material for the study was red blood cells and blood plasma of patients received in the dynamics – the acute period (during hospitalization); 3-4 weeks later – the phase of early convalescence with a non-smooth progression and moderate and heavy severity; the recovery period with a smooth progression and mild and moderate severity; 5-6 weeks later – the recovery period with a non-smooth progression and moderate and heavy severity. The spectrophotometric method was used to study lipid peroxidation (the concentration of diene conjugates, TBA-reactive substances) in the blood plasma and components of the antioxidant support system (the content of reduced glutathione; the activity of glutathione reductase, glutathione peroxidase, and catalase) in red blood cells.Results. It was determined that moderate and heavy pseudotuberculosis forms prevail in hospitalized children, the disease progression in 35.2% of them was non-smooth; lipid peroxidation products accumulate in the blood plasma and the concentration of reduced glutathione decreases in red blood cells during the acute period of pseudotuberculosis in all children relative to the parameters in the control group. In the period of early convalescence an imbalance in the functioning of antioxidant enzymes of red blood cells, as well as the accumulation of TBA-reactive substances and a decrease in the content of reduced glutathione were observed in patients with moderate and heavy pseudotuberculosis.Conclusion. The impact of the imbalance of pro-/antioxidants on the formation of predominantly moderate and heavy pseudotuberculosis in children is shown. Prognostic criteria for the development of a non-smooth progression of pseudotuberculosis are a high level of lipid peroxidation products in the blood plasma, no normalization in values of glutathione system components and the activity of erythrocyte catalase during early convalescence.

Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.

A RARE CAUSE OF NEONATAL CYANOSIS - CONGENITAL METHEMOGLOBINEMIA — A CASE REPORT

Cyanosis can occur at any age but it is a great challenge when it is in the newborn. Multiple causes have been established, and it usually is an ominous sign, especially when it occurs with neonatal sepsis or cyanotic congenital heart disease. Cyanosis caused by abnormal forms of hemoglobin can be life threatening, and early recognition is mandatory to prevent unnecessary investigations and delay in management. Abnormal hemoglobin newborn screening is a useful tool for the diagnosis. Acquired methemoglobinemia caused by environmental oxidizing agents, is common, but congenital deficiency of the innate reducing enzyme is so rare that only a few cases are documented in the medical literature around the world. Here I am presenting a case of a neonate with cyanosis as a result of congenital deficiency of cytochrome b5 reductase enzyme. This neonate was found to have cyanosis and appeared blue at newborn follow-up visit. Sepsis, congenital heart disease, prenatal administration, and ingestion of oxidant dyes were excluded as causes of the cyanosis by history, examination and appropriate tests. Chocolate discoloration of arterial blood provided a clue to the diagnosis. A normal newborn screen and hemoglobin electrophoresis made the diagnosis of hemoglobin M unlikely as the cause of the methemoglobinemia (Hb A 58.9%, A2 1.9%, and F 39.2%). Red blood cell enzyme activity and DNA analysis revealed a homozygous form of the cytochrome b5 reductase enzyme deficiency. She responded very well to daily methylene blue and ascorbic acid administration, and she has normal growth and developmental parameters, although she shows an exaggerated increase in her methemoglobin level.

Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency Among Children in Eastern Saudi Arabia.

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common red blood cell enzyme deficiency worldwide. The disease is widely distributed in regions where malaria is prevalent, affecting mostly males because the enzyme is inherited as an X-link recessive pattern. In Saudi Arabia, we lack newborn screening (NBS) for G6PD deficiency, despite early reports about high prevalence.MethodsThis is a 10-year retrospective study of children who were screened for G6PD deficiency during their hospitalization between January 2008 to December 2017. The test was carried out using a qualitative fluorescence test suitable for mass screening to determine the prevalence of G6PD deficiency among the admitted children between 0 and 14 years of age.ResultsA total of 48,889 patients were screened which included 27,634 (56.5%) males and 21,255 (43.5%) females with a mean age of 1.93 + 3.98 years. The overall prevalence of G6PD deficiency was 25%, whereas it was 33.8% in the male subset and 13.2% in the female subset. Male sex was significantly correlated with G6PD deficiency. A total of 25,628 newborns were screened, with 14,219 (55.5%) males and 11,409 (44.5%) females, who had a G6PD deficiency prevalence of 18.8%. There was a G6PD deficiency prevalence of 26% in males and 9.9% in females.ConclusionThe present study confirms the high prevalence of G6PD deficiency in our community. Therefore, we need to establish an NBS program to screen for G6PD deficiency in order to prevent neonatal hyperbilirubinemia encephalopathy, avoidable hemolytic episodes, and to increase awareness among health practitioners.

Impact of Next-Generation Sequencing on the Diagnosis and Treatment of Congenital Anemias.

Congenital anemias are a wide spectrum of diseases including hypoproliferative anemia syndromes, dyserythropoietic anemias, sideroblastic anemias, red blood cell membrane and enzymatic defects, hemoglobinopathies, and thalassemia syndromes. The various congenital anemia syndromes may have similar clinical and laboratory presentations, making the diagnosis challenging. The traditional work-up, which includes a complete blood count, blood smears, bone marrow studies, flow cytometry, and the osmotic fragility test, does not always lead to the diagnosis. Specialized tests such as red blood cell enzyme activity and ektacytometry are not widely available. In addition, red blood cell transfusions may mask some of the laboratory characteristics. Therefore, genetic testing is crucial for accurate diagnosis of patients with congenital anemias. However, gene-by-gene testing is labor intensive because of the large number of genes involved. Thus, targeted next-generation sequencing using custom-made gene panels has been increasingly utilized, with a high success rate of diagnosis. Accurate genetic diagnosis is important for determining specific therapeutic modalities, as well as for avoiding splenectomy when contraindicated. In addition, molecular diagnosis can allow for genetic counseling and prenatal diagnosis in severe cases. We suggest a work-up scheme for patients with congenital anemias, including early incorporation of targeted next-generation sequencing panels.

A Rare Case of Coexistence of Homozygous β-Thalassaemia and Glucose 6 Phosphate Dehydrogenase Deficiency

β-thalassaemia is one of the most prevalent autosomal disorders worldwide. Mutations/deletions in globin gene underlie deficiencies in Haemoglobin (Hb) production, which can interfere with oxygen delivery by Hb, resulting in thalassaemias causing anaemias with a wide range of disease severity. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is a genetic abnormality resulting in inadequate amount of G6PD in the Red Blood Cells (RBCs). In patients with G6PD deficiency, the reduced or absent activity of the enzyme in RBCs causes premature haemolysis and symptomatic anaemia. The marked oxidative stress caused by homozygous β-thalassaemia is apparently incompatible with G6PD deficiency. Here, a rare case of six-month-old male child is described who presented with severe pallor hepato-splenomegaly and these two conditions co-existed in this patient.

Hereditary hemolytic anemia in newborns: clinical significance of genetic diagnosis

Hereditary hemolytic anemia (HHA) is a group of monogenic diseases arising from the increased destruction of circulating erythrocytes. HHA is caused by germline mutations in genes encoding components of the red blood cell membrane, hemoglobin, and enzymes. Comprehensive gene analyses have identified various HHA-associated germline defects. However, early HHA diagnosis can be difficult in newborns because they present with hydrops and severe jaundice, which require urgent blood transfusions. Considering neonatal physiological hemolysis and pediatric infection, we select efficient diagnostic procedures following the exclusion of "syndromic hemolytic diseases". Clinical sequencing is performed for atypical cases, although phenotypic and laboratory tests remain essential for the verification of pathogenicity when certain variants are identified. The diagnostic gene panel can also be useful for predicting prognosis and determining treatment options. Although transfusion-dependent adult patients with HHA are rare in Japan, their management remains challenging. Clinical trials of new drugs and genetic controls are ongoing in other countries. However, the long-term management of a small group of patients with severe HHA must still be addressed in Japan. Here, we review the strategy and clinical significance of using genetic diagnostic methods for HHA in newborns.

Insights into the ameliorative effect of oleic acid in rejuvenating phenylhydrazine induced oxidative stress mediated morpho-functionally dismantled erythrocytes

Phenylhydrazine (PHZ), an intermediate in the synthesis of fine chemicals is toxic for human health and environment. Despite of having severe detrimental effects on different physiological systems, exposure of erythrocytes to PHZ cause destruction of haemoglobin and membrane proteins leading to iron release and complete haemolysis of red blood cells (RBC). Involvement of oxidative stress behind such action triggers the urge for searching a potent antioxidant. The benefits of consuming olive oil is attributed to its 75% oleic acid (OA) content in average. Olive oil is the basic component of Mediterranean diet. Hence, OA has been chosen in our present in vitro study to explore its efficacy against PHZ (1 mM) induced alterations in erythrocytes. Four different concentrations of OA (0.01 nM, 0.02 nM, 0.04 nM and 0.06 nM) were primarily experimented with, among which 0.06 nM OA has shown to give maximal protection. This study demonstrates the capability of OA in preserving the morphology, intracellular antioxidant status and the activities of metabolic enzymes of RBCs that have been diminished by PHZ, through its antioxidant mechanisms. The results of the present study firmly establish OA as a promising antioxidant for conserving the health of erythrocyte from PHZ toxicity which indicate toward future possible use of OA either singly or in combination with other dietary components for protection of erythrocytes against PHZ induced toxic cellular changes.

Relationship of Host Genetic Factors with Severe Malaria in Nigerian Children

Host genetic factors that influence the outcome of Plasmodium falciparum malaria infection are not fully understood. These include alpha-thalassemia, sickle cell disease, glucose-6-phosphate dehydrogenase (G6PD) deficiency and cytochrome b5 reductase 3 (CYB5R3) T117S and ferroportin Q248H polymorphisms. G6PD, an X-linked gene, encodes the sole enzyme in red blood cells that produces NADPH for protection from reactive oxygen species. Hemi- or homozygosity for G6PD A+ (G6PD c. 376G) is an African specific polymorphism with reduced activity (PMID:26633385) but no apparent phenotype; hemi- or homozygosity for G6PD A- (G6PD c 202A/376G) is associated with both decreased activity and increased risk for oxidant-induced hemolysis (PMID:27040960). A Malaria Genomic Epidemiology Network (MalariaGEN) study with almost 30,000 participants reported that G6PD A- increases the risk for severe malarial anemia (PMID:25261933). CYB5R3 in red blood cells transfers electrons from NADH to cytochrome b5, which in turn converts methemoglobin to hemoglobin. The African-specific CYB5R3 T117S variant, is not associated with methemoglobinemia (PMID:9048929) but may afford some protection from malarial anemia in Zambian children. A prevalent African-specific Q248H mutation of the iron exporter, ferroportin, may protect from high malarial parasitemia (PMID: 29599243). We genotyped 212 Nigerian children (age 4 months - 16 years) for these genetic factors: 117 were asymptomatic children who were studied in the community and 95 were children who were studied at the time of admission to hospital with severe malaria (severe anemia and/or cerebral malaria). We isolated DNA from dried blood spots and crude DNA lysate was used for Taqman genotyping to screen for G6PD A+, G6PD A-, FPN Q248H, sickle cell disease and CYB5R3 T117S and multiplex PCR was used to identify the 3.7 kb and 4.2 kb α-globin gene deletions. The clinical characteristics of the two groups of children are shown in Table 1. Children with severe malaria were significantly younger than the children from the community. The gene frequency of CYB5R3 T117S was 0.303, of hemoglobin S was 0.094, and of FPN Q248H was 0.062. The prevalences of hemizygous and homozygous G6PD A+ and A- were 13.9% and 16.0%, respectively. In addition, the prevalences of single and double alpha globin gene deletions were 34.7 % and 11.7%, respectively. Table 2 compares the prevalence of these genotypes in asymptomatic children versus those admitted with severe malaria. Alpha-thalassemia tended to be more prevalent in the asymptomatic children while ferroportin Q248H and G6PD A- were more prevalent in the children with severe malaria. Some genes seemed to have heterogenous associations: sickle trait and homozygous CYB5R3 T117S tended to be more prevalent in the asymptomatic children while hemoglobin SS and heterozygous T117S tended to be more prevalent in the children with severe malaria. In a logistic regression model that adjusted for age, alpha thalassemia (odds ratio 0.6, P=0.038), hemoglobin S trait (odds ratio 0.5, P=0.203), and CYB5R3 T117S homozygous state (odds ratio 0.5, P=0.211) independently decreased the risk of severe malaria, while ferroportin Q248H (odds ratio 5.1, P=0.008) and G6PD deficiency (1.6, P=0.049) independently increased the risk of severe malaria. In conclusion, our results suggest that common genetic factors affect the risk of severe malaria in children. Research to illuminate the mechanisms of these effects may enhance our understanding of the pathogenesis of severe malaria and point to novel preventive and therapeutic measures. Disclosures Gordeuk: Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding.

PS1202 DIFFERENTIAL DIAGNOSIS OF CONGENITAL HEMOLYTIC ANEMIA OF NEONATES AND INFANTS IN JAPAN

Background:As a reference center, we analyze erythrocyte membranes, enzymes, and hemoglobin for congenital hemolytic anemia (CHA) in cases that are not diagnosed using ordinary laboratory examinations, and we introduce genetic testing using a hemolytic anemia‐related gene panel. We analyzed 136 cases from 2016 to 2017, including 36 patients under 1 year of age and 100 patients aged 1 year and over. Consequently, a definitive diagnosis was obtained for 84% of patients aged 1 and over but for only 72% of patients under 1 year of age.Aims:We aimed to summarize the final diagnosis for hemolytic anemia in patients less than 1 year of age analyzed over 3 years from 2016 to 2018 and discuss the common clinical features and laboratory data of undiagnosed cases.Methods:We analyzed 58 infantile CHA casesusing the following laboratory tests: flow‐cytometric osmotic fragility (FCM‐OF) test, eosin 5’‐maleimide binding test, assay of 15 red blood cell (RBC) enzymes, reduced glutathione content, and isopropanol stability test for unstable hemoglobinopathy. For the cases with a difficult diagnosis, genetic analysis with target‐captured sequencing (TCS) using a gene panel of 74 genes related to CHA was carried out.Results:Among 58 infants with CHA, we diagnosed 30 cases (52%); there were 18 cases of hereditary spherocytosis (HS), 2 cases of hereditary elliptocytosis (HE), 1 case of hereditary pyropoikilocytosis, 4 cases of erythro‐enzymopathies, and 2 cases of unstable hemoglobinopathies. Among the 28 undiagnosed cases, 14 showed an elevated percentage of residual RBCs in the FCM‐OF test. These cases also showed abnormal RBC morphologies such as target cells, stomatocytes, and/or pyknocytes. We performed TCS for 4 of the 14 cases, but none harbored mutations such as PIEZO1 or KCNN4. Of the 7 cases, which could be followed up to at the age of one year, all recovered from CHA within a year.Summary/Conclusion:Mild hemolytic anemia accompanied by stomatocytes and/or target cells is a typical clinical feature of dehydrated hereditary stomatocytosis (DHSt) in adults. We previously showed that FCM‐OF clearly discriminated DHSt from HS or HE (Utsugisawa et al., Blood 2017;130:929). In this study, approximately half of the infants with undiagnosed CHA showed similar RBC morphology and higher osmotic resistance with no causative gene mutations. We noticed that clinical pictures of these infants resembled the description of infantile pyknocytosis (IP), which is a transient neonatal hemolytic anemia with the characteristic RBC morphology. As IP cases follow a relatively benign clinical course, we propose that FCM‐OF may be used as a biomarker for the tentative diagnosis of IP.

PS1203 HEREDITARY SPHEROCYTOSIS: SCREENING AND DIAGNOSTIC APPROACH IN A BELGIAN LABORATORY

Background: As a reference center, we analyze erythrocyte membranes, enzymes, and hemoglobin for congenital hemolytic anemia (CHA) in cases that are not diagnosed using ordinary laboratory examinations, and we introduce genetic testing using a hemolytic anemia-related gene panel. We analyzed 136 cases from 2016 to 2017, including 36 patients under 1 year of age and 100 patients aged 1 year and over. Consequently, a definitive diagnosis was obtained for 84% of patients aged 1 and over but for only 72% of patients under 1 year of age. Aims: We aimed to summarize the final diagnosis for hemolytic anemia in patients less than 1 year of age analyzed over 3 years from 2016 to 2018 and discuss the common clinical features and laboratory data of undiagnosed cases. Methods: We analyzed 58 infantile CHA casesusing the following laboratory tests: flow-cytometric osmotic fragility (FCM-OF) test, eosin 5’-maleimide binding test, assay of 15 red blood cell (RBC) enzymes, reduced glutathione content, and isopropanol stability test for unstable hemoglobinopathy. For the cases with a difficult diagnosis, genetic analysis with target-captured sequencing (TCS) using a gene panel of 74 genes related to CHA was carried out. Results: Among 58 infants with CHA, we diagnosed 30 cases (52%); there were 18 cases of hereditary spherocytosis (HS), 2 cases of hereditary elliptocytosis (HE), 1 case of hereditary pyropoikilocytosis, 4 cases of erythro-enzymopathies, and 2 cases of unstable hemoglobinopathies. Among the 28 undiagnosed cases, 14 showed an elevated percentage of residual RBCs in the FCM-OF test. These cases also showed abnormal RBC morphologies such as target cells, stomatocytes, and/or pyknocytes. We performed TCS for 4 of the 14 cases, but none harbored mutations such as PIEZO1 or KCNN4. Of the 7 cases, which could be followed up to at the age of one year, all recovered from CHA within a year. Summary/Conclusion: Mild hemolytic anemia accompanied by stomatocytes and/or target cells is a typical clinical feature of dehydrated hereditary stomatocytosis (DHSt) in adults. We previously showed that FCM-OF clearly discriminated DHSt from HS or HE (Utsugisawa et al., Blood 2017;130:929). In this study, approximately half of the infants with undiagnosed CHA showed similar RBC morphology and higher osmotic resistance with no causative gene mutations. We noticed that clinical pictures of these infants resembled the description of infantile pyknocytosis (IP), which is a transient neonatal hemolytic anemia with the characteristic RBC morphology. As IP cases follow a relatively benign clinical course, we propose that FCM-OF may be used as a biomarker for the tentative diagnosis of IP.

PB1973 CONTRIBUTION OF NGS IN THE DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS: CASE REPORT

Background:Hereditary spherocytosis (HS) is the most frequent cause of non‐immune haemolytic anaemia (HA) among individuals of Northern European ancestry; its estimated prevalence is 1:2000. HS results from various molecular defects leading to decreased production or abnormality of one or more red blood cell (RBC) membrane proteins i.e., ankyrin, protein 4.2, band 3 and spectrin. The majority of HS cases (75%) are inherited in an autosomal dominant manner. Especially because of the spherical shape, RBCs are trapped in the spleen and have a shortened life span. Among other factors, according to the importance of the deficiency or protein abnormality it conducts to a clinical expression that may range from asymptomatic to severe chronic HA. Most often a phenotypic diagnosis can be made but next‐generation gene sequencing (NGS) has a place in difficult cases i.e. unsolved cases of HA, transfusion prior to diagnosis and discrepancy between clinical phenotype and diagnosis.Aims:We report here the case of a 18‐year‐old patient with a suspected HS, prominent splenomegaly and unknown personal or family history.Methods:Case report:The patient complained of fatigue for several months and grade 2 dyspnea. In the past, he repeatedly presented with biliary colic pain. He apparently had no personal or family medical history. Two years ago, he nevertheless had jaundice secondary to haemolytic anaemia. The evolution was spontaneously favourable and no further investigation was undertaken. He has a twin brother and a 20‐year‐old sister, both in good health. The clinical examination found a prominent splenomegaly. Haemoglobinopathy and most frequent RBC enzyme deficiencies were excluded. Given the high clinical suspicion of HS, his parents and siblings (although asymptomatic) were screened for HS.Results:The main biological data are reported in the Table 1.Molecular genetic analyses were performed: NGS targeted sequencing panel, containing panel 40 genes associated to haemolytic anemia, and confirmation by Sanger identified a nonsense mutation on the cytoplasmic domain of Band3. The mutation is novel and drastic; it causes a stop codon at 75 residues. The mutation is transmitted by the mother. NGS targeted sequencing analysis did not demonstrate any other abnormalities that could explain the phenotypic variability observed in the propositus and his mother.Given the profound anaemia associated to the voluminous spleen (main diameter of 25 cm by CT‐scan evaluation) and given the presence of symptomatic gallstones, the patient underwent a splenectomy and a cholecystectomy. After the procedure, he recovered from fatigue and the haemoglobin level returned to normal with no signs of residual haemolysis.Summary/Conclusion:This case reports a HS related to a new nonsense mutation in SLC4A1. The transmission is autosomal dominant but the penetrance is heterogeneous within the same family.image

PB1967 HEREDITARY ELLYPTOCYTOSIS ASSOCIATED WITH HETEROZYGOTE PYRUVATE KINASE AND GLUCOSE‐6‐PHOSPHATE DEHIDROGENASE MUTATIONS. A CASE REPORT AND FAMILY STUDY

Background:Hereditary elliptocytosis (HE) is a red blood cell (RBC) defect characterized by an abnormal shape and mild or moderate chronic haemolytic anaemia. Most cases of HE are due to missense mutations in the RBC membrane skeleton α‐spectrin (SPTA1) gene that encodes α‐spectrin chains, a critical component that maintains cell shape and membrane elasticity. Families carrying α‐HE alleles are in general symptomless or exhibit a mild to moderate haemolysis whereas the more severely affected individuals have been observed in hereditary pyropoikilocytosis (HPP).Aims:The main objective of our task is to provide a fast and efficient diagnosis of Hereditary Haemolytic Anaemia (HHA) using Next Generation Sequencing (NGS).Methods:A NGS analysis including 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) was performed using an Illumina HiSeq2000 device.Results:Two identical twins were prematurely born at 34 weeks of gestation and their parents, a 41‐year‐old women and a 45‐year‐old man were healthy and did not exhibit medical history of interest. After the 10th day of life, an acute severe haemolytic crisis, of unknown origin, appeared in both premature siblings (Hb:70 g/l, reticulocytes:161,6x10^9/l; and Hb 81 g/L, reticulocytes 151x109/L respectively). RBC morphology exhibited unspecific characteristics, and in order to seek for a diagnosis, both parents were evaluated. In the mother, a slight well‐compensated haemolysis (Hb:110 g/l, reticulocytes:102 x109/l) with about 25% of elliptocytes and ovalocytes was found, whereas the father was normal. After NGS study, four different variants were identified in the mother: a) Two α‐spectrin variants in a double heterozygous condition: SPTA1 c.779T&gt;C (p.Leu260Pro), and SPTA1 (c.6531‐12GC&gt;A) α‐LELY), b) A pyruvate kinase gene (PKLR) variant: c.829G&gt;A (p.Glu277Lys); and c) A glucose‐6‐phosphate dehydrogenase (G6PD) A gene variant: c.968T&gt;C (p.Leu323Pro).The SPTA1 (c.779T&gt;C), PKLR (c.829G&gt;A) and G6PD (c.968T&gt;C) mutations, in heterozygous condition, were also found in both children. No gene variants were found in the father.Summary/Conclusion:We present a typical case of symptomless HE in a woman that was studied with NGS due to the severe neonatal haemolytic crisis found in her both premature siblings. Despite this NHC was initially attributed to the mother's disease (HE), the NGS allowed to found two additional mutations of RBC enzyme genes (PK and G6PD) in both siblings but also in the mother that may be the cause of the unexpected crisis. However, despite in the mother the SPTA1 c.779T&gt;C mutation seems to be aggravated by the coexistence, in trans, of the α‐LELY allele (αHE/αLELY diplotype), this was not the case in the siblings because they inherited the α‐LELY allele but not the elliptogenic mutation. Accordingly, after the NGS study it has to be assumed that both proband's sons are at risk allowing to suggest that G6PD deficiency as the mechanism of the NHC observed in both of themThe present report is an example of the utility of NGS to distinguish potential genetic mechanisms of HHA, and to find unexpected genetic variants or mutations in patients with slight or moderate phenotypic expression of the disease. This provides a new challenge to investigate non‐phenotypically suspected complex genetic situations, and likewise to the improvement of the diagnosis and genetic counselling of patients with rare anaemias.image

Molecular diagnostic update in hereditary hemolytic anemia and neonatal hyperbilirubinemia.

Hereditary hemolytic anemia (HHA) is a group of genetically and phenotypically heterogeneous disorders characterized by premature destruction of red blood cells (RBCs) with clinical manifestations ranging from asymptomatic to marked hemolytic anemia. There are three main categories of HHA: (a) RBC membrane defects; (b) hemoglobinopathies/thalassemias; and (c) RBC enzyme deficiencies. Hyperbilirubinemia is a frequent consequence of hemolytic anemia and can lead to bilirubin-associated neurotoxicity in neonates and to jaundice, and formation of gall stones in adults. Hyperbilirubinemia can also be caused by impaired bilirubin conjugation due to polymorphisms and mutations in genes involved in bilirubin metabolism (eg, UGT1A1). Neonates with HHA and co-inherited variants impairing bilirubin conjugation are at increased risk of bilirubin-associated toxicity. Prior to the advent of next-generation sequencing (NGS), molecular diagnosis of these disorders was limited to targeted single gene Sanger sequencing. However, NGS is making its way into the standard diagnostic workup of complex and multigene disorders like HHA. This review will focus on the molecular updates of HHA with particular focus on the neonatal and pediatric population.

Melatonin chelates iron and binds directly with phenylhydrazine to provide protection against phenylhydrazine induced oxidative damage in red blood cells along with its antioxidant mechanisms: an in vitro study

Oxidative stress is an important causative factor for a number of diseases. Phenylhydrazine (PHZ) is a widely accepted model for studying hemolytic anemia by induction of oxidative stress. In the present study, goat red blood cells (RBCs) were incubated in vitro with PHZ (1mM) to generate oxidative stress. To test whether melatonin exhibits protective effects on PHZ induced RBC damage and to explore its potential molecular mechanisms, different concentrations of melatonin (5, 10, 20 and 40 nmoles/ml) were also included. PHZ caused altered profiles on biomarkers of oxidative stress and antioxidative as well as glucose metabolic enzymes in RBCs. These alterations indicated a development of oxidative stress. Melatonin at a concentration of 40 nmoles/ml provided optimal protection against all alterations induced by PHZ. The important cellular membrane proteins, including spectrin and actin, were also damaged by PHZ and this led to RBC deformation similar to that of observed in severe β-thalassaemia; the RBC deformation was also prevented by melatonin. Binding profiles of melatonin with PHZ and ferrous iron indicated favorable binding of melatonin with both of them, respectively. Thus, in addition to the direct antioxidant and free radical scavenging capability, melatonin also inhibited iron overloading by chelating iron and binding with the PHZ. This action of melatonin further reduces free radical generation. Based on the results, melatonin may provide therapeutic relevance to ß-thalassemia and other hemolytic RBC disorders involving oxidative stress.

Oxidative Stress Caused by an SOD1 Deficiency Triggers the Accumulation of Oxidatively Modified Carbonic Anhydrase II in Erythrocytes

Red blood cells (RBCs) are continuously exposed to reactive oxygen species (ROS) that are largely produced from intrinsic sources such as the oxidation of hemoglobin. Since superoxide dismutase 1 (SOD1) is the sole superoxide-scavenging enzyme in RBCs, a deficiency leads to the development of hemolytic anemia in mice, suggesting that the underlying mechanism involves a massive, oxidative stress-induced destruction of RBCs. We recently reported a decreased proteasomal function and the accumulation of ubiquitinated proteins in RBCs in SOD1-knockout (KO) mice. Because proteasomes are responsible for the degradation of both ubiquitinated proteins and oxidatively-modified (oxidized) proteins without ubiquitination, their malfunction results in the accumulation of these proteins. In the current study, we examined the issue of how elevated ROS are involved in the destruction of RBCs and the onset of anemia from the point of view of the accumulation of oxidized proteins. The findings indicate that carbonic anhydrase II (CAII) was the major protein within RBCs that was oxidized and that high levels had accumulated in SOD1-KO RBCs. Using purified CAII, we demonstrated that oxidative modification decreased its enzymatic activity in a ROS-dependent manner. In addition, the oxidized CAII molecules appeared to be degraded by proteasomes in RBCs. Based on these findings, we conclude that oxidative stress caused by an SOD1 deficiency disrupts the scavenging activity of proteasomes and accelerates the accumulation of oxidized CAII, leading to RBCs having a shortened life span.