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Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 μg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.

Key Points We show that the novel KCNN4 variant p.S314P is a gain-of-function mutation but is less severe than the previously reported p.R352H variant. The clinical heterogeneity, blurred symptoms, and absence of specific diagnostic markers make the diagnosis of Gardos channelopathy challenging.

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and β-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6–1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

Rasburicase is a recombinant urate oxidase enzyme indicated for tumor lysis syndrome, a potential life-threatening oncologic emergency that occurs most commonly during initial chemotherapy for hematological malignancies. As a result of the defects in the physiological antioxidant pathway, erythrocytes of patients with glucose-6-phosphate dehydrogenase deficiency are not protected against the oxidizing stress exerted by hydrogen peroxide generated with the administration of rasburicase. The authors report a 14-year-old patient, diagnosed with T-cell acute lymphoblastic leukemia, who developed methemoglobinemia and hemolytic anemia with low oxygen saturation after starting steroids, hyperhydratation, and rasburicase administration. The complications resolved with supportive therapy only.

Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias (CDA), and some forms of congenital hemolytic anemias. Among them pyruvate kinase deficiency (PKD) has been shown to develop iron overload also in absence of transfusions suggesting dyserythropoietic features. Moreover, severe forms can be misdiagnosed as CDA due to bone marrow abnormalities and ineffective erythropoiesis further supporting this evidences. The hormone erythroferrone (hERFE) is produced by erythroblasts in response to erythropoietin (EPO), and acts by suppressing hepcidin, thereby increasing iron absorption and mobilisation for erythropoiesis demand. The ERFE-hepcidin axis seems to play a crucial role in the pathogenesis of these disorders; an increased erythroferrone release by immature erythroid cells results in hepcidin suppression and secondary iron overload that could finally results in ineffective erythropoiesis and anemia. To investigate the pathophysiological basis of iron overload in PKD, we analysed the levels of hERFE, EPO, hepcidin, and soluble transferrin receptor (sTFR) in a large group of 41 PKD patients equally distributed by gender, age and severity. The results were analysed in comparison with two groups of patients affected by hemolytic anemia with overt dyserythropoiesis (42 patients with CDA type II) and with congenital hemolytic anemia due to RBC membrane defects (51 patients with hereditary spherocytosis [HS]), respectively. Demographic, hematologic, and biochemical features of the three groups of patients are reported in the table. Among the PKD patients, 18/41 were 6 tx/yrs). Mean ferritin levels at the time of the study were 546 ng/ml (range 59-4990), 15/41 patients requiring chelation therapy for iron overload developed also in absence of transfusions. As expected, CDAII patients showed decreased hepcidin levels (3.74 ng/mL; n.v. 17.25, P This study provides the first analysis of the main regulators of systemic iron homeostasis in PK deficiency compared either with the model of a structural RBC defect (HS) or with the typical model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. These data provide evidence of the dyserythropoietic features of PK deficiency, underlining the need of accurate diagnosis and paving the way of novel therapeutic approaches in PK deficiency. Zaninoni A. and Russo R. equally contributed to the study Figure 1 Figure 1. Disclosures Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Barcellini: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria. Iolascon: Bluebird Bio: Other: Advisory Board; Celgene: Other: Advisory Board. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hereditary xerocytosis (HX) is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve.PIEZO1gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy.

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, the second reaction step of glycolysis. GPI deficiency, transmitted as an autosomal recessive trait, is considered the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase deficiency. Despite this, this defect may sometimes be misdiagnosed and only about 60 cases of GPI deficiency have been reported. GPI deficient patients are affected by chronic non-spherocytic hemolytic anemia of variable severity; in rare cases, intellectual disability or neuromuscular symptoms have also been reported. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far, about 40 causative mutations have been identified. We report the clinical, hematological and molecular characteristics of 12 GPI deficient cases (eight males, four females) from 11 families, with a median age at admission of 13 years (ranging from 1 to 51); eight of them were of Italian origin. Patients displayed moderate to severe anemia, that improves with aging. Splenectomy does not always result in the amelioration of anemia but may be considered in transfusion-dependent patients to reduce transfusion intervals. None of the patients described here displayed neurological impairment attributable to the enzyme defect. We identified 13 different mutations in the GPI gene, six of them have never been described before; the new mutations affect highly conserved residues and were not detected in 1000 Genomes and HGMD databases and were considered pathogenic by several mutation algorithms. This is the largest series of GPI deficient patients so far reported in a single study. The study confirms the great heterogeneity of the molecular defect and provides new insights on clinical and molecular aspects of this disease.

Pyruvate kinase (PK) deficiency is the most common defect of the glycolytic pathway leading to congenital hemolytic anemia. We present the case of an 18-year-old boy with chronic nonspherocytic hemolytic anemia, who had remarkable sensitivity to heat. Moreover, the patient showed clinical impairment in the last year. For this reason, we excluded the immunologic or infectious nature (malaria, babesia), which may play a role in the worsening of anemia. Red blood cell enzyme assay showed the presence of a significant increase in other enzyme activities, except for PK, suggesting a PK deficiency in the patient. The molecular analysis of the PK-LR gene revealed the presence of a novel homozygote missense mutation (c.581G>C, p.Arg194Pro). The mutant enzyme displayed heat instability. In addition, we analyzed bilirubin uridine diphosphate (UDP)-glucuronosyltransferase 1A1 gene that revealed a heterozygous state ([TA]6/[TA]7). After a clear diagnosis of PK deficiency, the patient underwent splenectomy.

BACKGROUND Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS RBC antibody–bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.

Background The laboratory diagnosis of hereditary spherocytosis commonly relies on NaCl-based or glycerol-based red cell osmotic fragility tests; more recently, an assay directly targeting the hereditary spherocytosis molecular defect (eosin-5′-maleimide-binding test) has been proposed. None of the available tests identifies all cases of hereditary spherocytosis.Design and Methods We compared the performances of the eosin-5′-maleimide-binding test, NaCl-osmotic fragility studies on fresh and incubated blood, the glycerol lysis test, the acidified glycerol lysis test, and the Pink test on a series of 150 patients with hereditary spherocytosis grouped according to clinical phenotype and the defective protein, with the final aim of finding the combination of tests associated with the highest diagnostic power, even in the mildest cases of hereditary spherocytosis.Results The eosin-5′-maleimide-binding test had a sensitivity of 93% and a specificity of 98% for detecting hereditary spherocytosis: the sensitivity was independent of the type and amount of molecular defect and of the clinical phenotype. The acidified glycerol lysis test and Pink test showed comparable sensitivity (95% and 91%). The sensitivity of NaCl osmotic fragility tests, commonly considered the gold standard for the diagnosis of hereditary spherocytosis, was 68% on fresh blood and 81% on incubated blood, and further decreased in compensated cases (53% and 64%, respectively). The combination of the eosin-5′-maleimide-binding test and acidified glycerol lysis test enabled all patients with hereditary spherocytosis to be identified. The eosin-5′-maleimide-binding test showed the greatest disease specificity.Conclusions Each type of test fails to diagnose some cases of hereditary spherocytosis. The association of an eosin-5′-maleimide-binding test and an acidified glycerol lysis test enabled identification of all patients with hereditary spherocytosis in this series and, therefore, represents a currently effective diagnostic strategy for hereditary spherocytosis including mild/compensated cases.

We report the hematological, biochemical, and molecular characteristics of a new defective adenylate kinase (AK) variant associated with chronic hemolytic anemia. The propositus was a 3-year-old girl of southern Italian origin with a history of severe anemia and occasional need for blood transfusion. The study of the most important red cell enzymes revealed low AK activity (22% of normal) in the propositus and intermediate values in the parents. The sequence of erythrocyte AK-1 gene showed a new homozygous mutation (delG138) determining a frameshift and a premature stop at codon 91.

Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency.Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed.The molecular characterization of the PKLR gene showed a heterozygous mutation 994GA (Gly332Ser) associated with the promoter substitution -148CT, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS.The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype.

Background Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency. Methods Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed. Results The molecular characterization of the PKLR gene showed a heterozygous mutation 994G > A (Gly332Ser) associated with the promoter substitution -148C > T, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS. Conclusions The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype.

The Gardos channel (KCNN4) is a Ca2+ sensitive, intermediate conductance, K+ selective channel abundant in several tissues including red blood cells (RBC) where it is involved in cell volume regulation (Hoffman et al. 2003, Cahalan et al. 2015). Hereditary dehydrate stomatocytosis (DHSt) is characterized by mild to moderate congenital hemolytic anemia with reticulocytosis and splenomegaly associated with RBC dehydration. Affected RBC are characterized by a nonspecific cation leak, reflected in elevated Na+ content, decreased K+, elevated MCHC and MCV, and decreased osmotic fragility. The definitive diagnosis of DHSt is made by osmotic gradient ektacytometry, which shows a leftward shift of the bell-shaped curve. Heterozygous mutations associated with delayed channel inactivation have been identified for PIEZO1 (Zarychanski et al. 2012), although there are patients lacking a mutation in this gene. Differential diagnosis in hereditary stomatocytosis is important because in DHSt patients, splenectomy is contraindicated due to increased risk of thromboembolic complications. We studied a 40 year-old Italian man, affected since infancy by chronic hemolyitc anemia (Hb 7-9 g/dL, reticulocytes 10%). The unrelated parents and three siblings were healthy with normal hematologic parameters. He was occasionally transfused until splenectomy, performed at the age of 11 years. After splenectomy Hb levels maintained around 10 g/dL, thrombotic events did not occur. Increased ferritin levels (>1000 ng/ml) and moderate iron overload required chelation therapy. Peripheral blood smear showed anysopoikilocytosis with stomatocytes (13%) echinocytes (7%) schistocytes (6%) ellyptocytes (5%) spherocytes (4%). Bone marrow evaluation revealed erythroid hyperplasia with some dyserytrhopoietc changes, in particular binucleated erythroblasts. Eosin 5 maleimide (EMA) binding test was negative; despite the high number of stomatocytes in peripheral blood, the osmotic gradient ektacytometry curve was not suggestive for hereditary xerocytosis. The proband's first daughter, born at term after an uneventful pregnancy, presented with severe anemia (Hb 6.1 g/dL) requiring RBC transfusion at birth and at 4 months; at the age of 1 year she displayed a clinical pattern similar to the one of the father, with median Hb levels of 9 g/dL and no need of transfusions. To uncover the underlying etiologies, all the family members underwent whole exome sequencing. Only one candidate gene, the KCNN4 could be identified: a heterozygous missense mutation (c.1055G>A, p.R352H) was detected as a de novo mutation in the proband, and dominantly transmitted to the daughter. The mutation falling in exon 7 involved in the calmodulin binding domain was predicted to be pathogenic by in silico analysis. Sanger sequencing confirmed the presence of the mutation. Very recently (July 2015), the same mutation in the KCNN4 gene was described by other groups (Rapetti-Mauss et al. 2015; Andolfo et al. 2015). In heterologous expression systems it could be shown that the Ca2+ sensitivity of the mutated Gardos channel was increased. We performed single cell experiments, including patch-clamp recordings in RBCs and precursor cells, Ca2+ fluorescence imaging as well as tracer flux experiments in RBCs of the above described patients and healthy controls to get a mechanistic link between the Gardos channel mutation and the cellular symptoms. Despite the presence of a single mutation in Gardos channel alone, pathological alterations in function of multiple ion transport systems were revealed during these studies. Our findings together with recent reports (Rapetti-Mauss et al. 2015; Andolfo et al. 2015; Glogowska et al. 2015) suggest that the prevalence of mutations in the Gardos channel is higher than previously thought. Therefore it is important to understand the mechanisms implicated by the mutation and to develop reliable diagnostic tools and treatment strategies. Disclosures No relevant conflicts of interest to declare.

We report the clinical, haematological and molecular characteristics of two triose phosphate isomerase deficient patients affected by haemolytic anaemia and neuromuscular impairment. The sequence of complete TPI gene showed the presence of two previously undescribed mutations: c.722 T>C (Phe240Ser) and c.28 insG; each of the two unrelated patients showed the new mutation in compound heterozygosity with the most common variant Glu104Asp. The association of Glu104Asp with c.28 insG resulted in a very severe clinical pattern.

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease characterized by ineffective erythropoiesis, hemolysis, erythroblast morphological abnormalities, and hypoglycosylation of some red blood cell (RBC) membrane proteins. Recent studies indicated that CDAII is caused by a defect disturbing Golgi processing in erythroblasts. A linkage analysis located a candidate region on chromosome 20, termed the CDAN2 locus, in the majority of CDAII patients but the aberrant gene has not so far been elucidated. We used a proteomic-genomic approach to identify SEC23B as the candidate gene for CDAII by matching the recently published data on the cytoplasmic proteome of human RBCs with the chromosomic localization of CDAN2 locus. Sequencing analysis of SEC23B gene in 13 CDAII patients from 10 families revealed 12 different mutations: six missense (c.40C>T, c.325G>A, c.1043A>C, c.1489C>T, c.1808C>T, and c.2101C>T), two frameshift (c.428_428delAinsCG and c.1821delT), one splicing (c.689+1G>A), and three nonsense (c.568C>T, c.649C>T, and c.1660C>T). Mutations c.40C>T and c.325G>A were detected in unrelated patients. SEC23B is a member of the Sec23/Sec24 family, a component of the COPII coat protein complex involved in protein transport through membrane vesicles. Abnormalities in this gene are likely to disturb endoplasmic reticulum (ER)-to-Golgi trafficking, affecting different glycosylation pathways and ultimately accounting for the cellular phenotype observed in CDAII.

We report the clinical and molecular characteristics of 6 new patients with recessive hereditary methemoglobinemia due to cytochrome b5 reductase deficiency. One patient was affected by Type-II disease with cyanosis and severe progressive neurological dysfunction, whereas the others displayed the benign Type-I phenotype. Methemoglobin levels ranged from 12.1% to 26.2% and cytochrome b5 reductase activity from 0 to 10% of normal. Eight different mutations were detected among the twelve mutated alleles identified, one splicing mutation, two stop codon, and five missense. Two mutations c. 82 C>T(Gln27STOP) and c. 136 C>T(Arg45Trp) are new. Prenatal diagnosis was performed in the family with Type-II disease.

Abstract 5270 Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells. The PGK1 gene is located on the Xq-13.1 chromosome, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in enzyme deficiency characterized at clinical level by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. Since the first description by Kraus et al, nearly 40 patients with PGK deficiency have been reported, 27 of them characterized at the DNA or protein level. To date, 20 different mutations with worldwide distribution have been described. The aim of the study was to characterize the molecular defect in an Italian patient affected by phosphoglycerate kinase deficiency. The patient, born from unrelated parents with negative family history of neurological defects, showed at birth neonatal jaundice. At the age of four years, in concomitance of an infective episode, he displayed hemolytic anemia (Hb 8.6 g/dL, reticulocytes 19%, unconjugated bilirubin 0.91 mg/dL, LDH 445 u/l, aptoglobin absent) and increased CPK values (2483U/L). The patient showed respiratory distress. The study of red cell glycolytic enzymes displayed a drastic reduction of PGK activity (41.8 UI/gHb ref. values 287–392). We examined again the patient at the age of 25 yrs in occasion of his sister's first pregnancy. The patient displayed compensated hemolytic anemia (Hb 14.1 g/dL, reticulocytes 6.6%) and severe myopathy. Sequencing analysis of the entire coding region and flanking intronic sequences of PGK1 gene showed the presence of a novel missense mutation c.1112 (ATA>AAA) responsible for amino acid substitution Ile371Lys. Although the mutation falls in the third last nucleotide of exon 9, it doesn't alter the splicing as confirmed by patient cDNA analysis that showed a normal transcript. The new mutation is located in a highly conserved region among species close to the ATP binding site and it was not found among the 100 normal alleles examined thus excluding the possibility of a polymorphism. Family study performed in the parents, the two healthy sisters and maternal uncle confirmed the X-linked transmission of the disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 5272 Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them. Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2). We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction. Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 4028 Poster Board III-964 CDAII, the most frequent type of congenital dyserythropoietic anemia family, is an autosomal recessive disease characterized by ineffective erythropoiesis, peripheral hemolysis, erythroblasts' morphological abnormalities and hypoglycosylation of some RBC membrane proteins. Recent studies indicated that CDAII is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts. Linkage analysis located the CDAII gene in a 5 cM region on chromosome 20 and several candidate genes have been excluded. We recently investigated the cytoplasmic proteome of human red blood cells (RBCs) using a combinatorial peptide ligand library as a capturing agent to amplify the signal of low- and very-low abundance proteins: 1578 proteins, most of them unexpected, were identified allowing a deep exploration of the RBC pathways (Roux-Dalvai, Mol Cell Proteomics, 2008). In this study we used a proteomic-genomic approach to identify the candidate gene for CDAII by matching the data on the cytoplasmic proteome of human RBC with the chromosomic localization of CDAN2 locus. The analysis of RBCs cytoplasmic proteome allowed us to identify 17 proteins codified by genes located in the chromosomic region between 20p11.23 and 20q11.23: SNX5, SEC23B, DTD1, NAT5, GINS1, BCL2L1, MAPRE1, CHMP4B, EIF2S2, AHCY, ACSS2, GSS, EIF6, CPNE1, EPB41L1, RPRD1B(C20orf77), TGM2. Most of them were excluded because found to be associated to other diseases, already excluded in CDAII by previous works, or because not related by function to CDAII. Among the remaining proteins we focus on SEC23B for its possible role in the endoplasmic reticulum-to Golgi trafficking and its localisation on 20p11, the region with the highest LOD-score in CDAII after the recent mapping of the markers on the current contigs (Denecke et al, Biochim Biophys Acta, 2009). The 20 exons and intronic flanking regions of SEC23B gene were analysed by direct sequencing in 16 CDA II patients from 13 families; 13 different mutations were detected among the 28 mutated alleles identified: 6 of them were missense, 2 frameshift, 1 splicing and 4 stop codon. All the missense mutations affected highly conserved aminoacids, and were not found in 100 normal alleles examined. Two of them (c.40C>T and c.325G>A) were detected in various unrelated patients. Despite all exons and flanking intronic regions were sequenced, one patient failed to show mutations and two cases displayed only one mutation, suggesting the possibility that a second gene could be involved in CDAII. Patients' data are summarised on the table (*,° = members of the same family). SEC23B is a member of the SEC23/SEC24 family, a component of COPII coat protein complex which is involved in protein trafficking through membrane vesicles. Very recently it has been shown that knockdown of Zebrafish SEC23B leads to aberrant erythrocyte development (Schwarz et al, Nat Genet, 2009). Even if the exact function of human SEC23B is not completely clarified, abnormalities in this gene are likely to disturb ER-to Golgi trafficking affecting different glycosylation pathways, and ultimately accounting for the cellular phenotype observed in CDA II. Case Sex Origin Hb g/dL Retics 10e9/L Band3 hypoglyc Mutations Exons Effects 1 F N Italy 9.7 160 Yes c.40 C>T/c.428 A>CG 2/5 Arg 14 Trp/Frameshift 2 F C Italy 11.4 44 Yes c. 1821 delT/? 16/? Frameshift/? 3 F Bolivia 9.9 61 Yes c.568 C>T/c.1808 C>T 5/16 Arg 190 STOP/Ser 603 Leu 4 M Rumania 9.8 102 Yes c.40 C>T/c.1660 C>T 2/14 Arg 14 Trp/Arg 554 STOP 5 M S Italy 10.4 nd Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 6 F C Italy 8.3 103 Yes c.40 C>T/Ivs6 +1g>a 2/Ivs6 Arg 14 Trp/Splicing 7 M C Italy 9.7 100 Yes c.1489C>T/c. 2101 C>T 13/18 Arg 497 Cys/Arg 701 Cys 8° F N Italy 9.2 121 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 9° M N Italy 11.3 115 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 10° F N Italy 11.7 63 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 11 F N Italy 11.6 104 Yes c.40 C>T/c.1043 A>C 2/9 Arg 14 Trp/Asp 348 Ala 12* M S Italy 11.9 90 Yes c.40 C>T/c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 13* F S Italy 7.8 61 Yes c.40 C>T/ c.649 C>T 2/6 Arg 14 Trp/Arg 217 STOP 14 F N Italy 9.5 105 Yes c.325 G>A/c.325 G>A 4/4 Glu 109 Lys/Glu 109 Lys 15 M N Italy 10.4 236 Yes c.367 C>T/? 5/? Arg 123 STOP/? 16 F S Italy 10.7 87 Yes ?/? ?/? ?/? Disclosures: No relevant conflicts of interest to declare.

Abstract 1981 Poster Board I-1003 Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of disorders characterized by ineffective erythropoiesis with prominent dysplastic features of the erythroid precursors in the bone marrow. Three main subtypes (I, II and III) have been identified, but several variants or atypical forms have been reported over the years. CDA are rarely associated with hydrops foetalis. In the past we described two cases presenting with hydrops foetalis and very severe anemia that were classified as atypical CDAs since they presented CDAII-like erythroblastic morphological features lacking other diagnostic CDAII markers. Very recently we and others (Bianchi et al, Human Mutat 2009, in press; Schwarz et al, Nat Genet 2009, in press) demonstrated that mutations in SEC23B gene, coding for a protein involved in the coat protein complex responsible for vesicle budding from the endoplasmic reticulum, cause CDA II. The aim of this work was to ascertain whether atypical CDAII-like forms presenting as hydrops foetalis could be caused by mutations in SEC23B gene and reclassified as CDAII. Two patients (Cantù-Rajnoldi et al, Br J Haematol 1997, 96: 530-3; Bianchi et al, Blood 1999, 94, suppl 1, 8b) from unrelated families with a history of intrauterine death of hydropic foetuses in previous pregnancies were referred at 20th week gestation following ultrasonic diagnosis of foetal hydrops and severe anemia (2.0 and 1.3 g/dL Hb respectively). In both cases intrauterine transfusions enabled the delivery of the babies. At birth both of them underwent extensive laboratory evaluation (including red cell metabolism and membrane proteins analysis) that was not informative on the causes of anemia. Ham test was repeatedly normal as for Western blotting analysis for GRP78 and glycoslylation pattern of red cell band 3. The bone marrow examination revealed the presence of 30 and 48% of bi- or multinucleated erythroblasts, some of whom presenting typical double outer membranes at transmission electron microscopy. Both babies became transfusion dependent. The 20 exons and intronic flanking regions of SEC23B gene were analyzed by direct sequencing. Both patients displayed mutations in SEC23B gene, in particular mutations c.325 G>A/ c.2101 C>T in patient 1 and c.325 G>A/ c.197G>A in patient 2 (Glu109Lys/ Arg701Cys and Glu109Lys/ Cys66Tyr respectively). c.325 G>A is the most frequent mutation so far described in CDAII and at homozygous level is usually associated with mild anemia. When found in compound heterozygosity with a second missense mutation as in these cases it may result in a very severe clinical pattern, although we cannot exclude that factors other than SEC23B mutations may contribute to worsening the clinical picture. In conclusion, SEC23B gene analysis allowed the correct classification of two very severe CDA cases associated with hydrops foetalis. This finding indicates that CDAII may result in intrauterine death and its frequency may therefore be underestimated. These cases may present as “atypical” for the lack of band3 hypoglycosylation likely due to the early sequestration of the more severely affected cells. Disclosures: No relevant conflicts of interest to declare.

We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin c. Mutation −73g>c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a>g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.

Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.

Recessive congenital methemoglobinemia (RCM) is caused by deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (cytb5r). The cytb5r gene (DIA1) is localized on chromosome 22q13-qter and contains nine exons; tissue-specific alternative transcripts originate 275-amino acid soluble and 300-amino acid membrane-bound isoforms. Cytb5r deficiency exists in two distinct clinical forms: in Type-I the enzyme deficiency is restricted to the red cell-soluble cytb5r isoform and results in cyanosis; in Type-II the enzyme defect involves both the soluble and membrane-bound isoforms, and severe neurological impairment is also present. More than 40 different mutations of DIA1 gene have been reported in RCM: disruptive mutations (stop, frameshift, splicing) are in general associated with the severe Type-II disease. We report the clinical, hematological and molecular characterisation 6 new patients affected by RCM (Table 1). Mutations reported in italic are new. The study of cytb5r enzyme showed a drastic reduction of activity in all patients. Parents displayed intermediate values characteristics of an heterozygous state. Mutation IVS5+2 t-c at the homozygous state was found in Type-II RCM (case 1) as already reported by Yilmaz et al (2005) in one patient; this variant is likely to cause alteration of mRNA splicing and subsequent absence of functional protein in all tissues, therefore accounting for the severe clinical pattern. V252M is the most common DIA1 gene mutation and has been reported in both Type-I and -II homozygous patients; the finding that this mutation in association with the new, very disruptive Gln28STOP mutation results in a benign phenotype (case 4), suggests that V252M is more likely to behave as a “Type-I” mutation. In case 6, in spite of cyanosis present since childhood, diagnosis of RCM Type-I was made in adult age because of polycythemia. In fact, significant compensatory elevation of hemoglobin concentration is sometimes observed in patients with congenital methemoglobinemia. Patient, (age) Type MetHb(%) Hb (g/dL) Hct (%) Cytb5r activity (IU/gHb) Mutations Effect 1 (8months) II 12.1 12.2 36.5 1.4 IVS5+2 t-c/IVS5+2 t-c Splicing/splicing 2 (2months) I 26.2 11.9 35.6 0.9 Cod.143 GGC-GAC/Cod.143 GGC-GAC Gly143Asp/Gly143Asp 3 (4months) I 12.4 16.6 49 1.7 Cod 218 CGA-TGA/Cod 105 GTG-ATG Arg218STOP/Val105Met 4 (1month) I 25.7 14.9 45 0 Cod 28 CAG-TAG/Cod. 252 GTG-ATG Gln28STOP/Val252Met 5 (1month) I Nd 12.2 35 1.9 Cod 46 CGG-TGG/Cod. 252 GTG-ATG Arg46Trp/Val252Met 6 (47years) I 23.7 18.6 55 0 cod 144 CCC-TCC/cod 252 GTG-ATG Pro144Ser/Val 252Met 0.5–1.5 12–16 39–49 15.4–23.1

Hereditary pyrimidine 5′ nucleotidase deficiency (P5′N) is the most frequent abnormality of the red cell nucleotide metabolism causing hereditary non-spherocytic hemolytic anemia. The disorder is characterized by mild-to-moderate hemolytic anemia associated with reticulocytosis and hyperbilirubinemia and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. P5′N-1 gene is localized on 7p15-p14; eighteen mutations have been so far identified in 27 unrelated families, 6 of them of Italian origin. The aim of this study is to describe the hematological, biochemical and molecular characteristics of two new Italian patients affected by P5′N deficiency. Case1: The propositus was a 37 yrs old woman of Northern Italian origin affected by chronic hemolytic anemia with Hb levels ranging from 8.2 to 10.5 g/dL. At the time of the study Hb was 8.4 g/dL, reticulocytes 300x109/L, unconjugated bilirubin 3.2 mg/dL. Peripheral blood smear examination showed basophilic stippling and purines/pyrimidines ratio (OD260/280) was decreased (1, ref. values 1.4 – 2.98). P5′N activity, measured by capillary electrophoresis, was undetectable. Molecular analysis of P5′N-1 gene showed the presence of a new homozygous deletion of two bp (ag) at the splice junction between intron 7 and exon 8, which probably results in a splicing alteration and in the absence of a functional protein. Case2: The propositus, a 37 yrs old woman of Northern Italian origin carrying the hemoglobin variant HbD Punjab, had an history of chronic hemolytic anemia since childhood; at the age of 14 yrs splenectomy and colecystectomy were performed. The patient needed blood transfusions because of exacerbation of anemia (Hb 4.7g/dL) during parvovirus B19 infection. Iron status parameters were increased requiring desferrioxamine treatment. At the time of the study Hb was 9.3 g/dL, reticulocytes 752x109/L, unconjugated bilirubin 13.3 mg/dL. Serum ferritin was 1980 mg/mL and transferrin saturation 115%. The propositus was found to be homozygous for Gilbert’s syndrome and heterozygous for mutation H63D of HFE gene. Basophilic stippling (6%) was observed in peripheral blood smear. Pur/pyr ratio was 0.8 and residual P5′N activity was 40% of normal. Complete sequencing of P5′N-1 gene showed the presence of the frameshift mutation ins GG710-711, already described in Italian and Turkish patients, and the new in-frame aminoacidic deletion of Gln 143.

Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyses the reversible isomerisation of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P), the second reaction step of glycolysis. GPI deficiency is the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far about 50 cases of GPI deficiency have been described, and 29 mutations have been reported at the nucleotide level: 25 missense, 2 nonsense and 2 splice site. The aim of this study is to describe the hematological, biochemical and molecular characteristics of two new families affected by GPI deficiency with chronic hemolytic anemia. Case1: The propositus was a 1 yrs old boy of Northern Italian origin with chronic hemolytic anemia and need for blood transfusion in concomitance of infectious episodes. At the time of the study Hb was 7.6 g/dL, reticulocytes 231x109/L, total bilirubin 1.4 mg/dL. The study of the most important red cell enzymes revealed a very low GPI activity (12.5% of normal). The parents were not consanguineous, and displayed intermediate GPI activity (66% of normal in both). The sequence of complete erythrocyte GPI gene revealed the presence of two new missense mutations: G145C (Gly49Arg) and C921A (Phe307Leu). Since both these aminoacids are conserved in eukaryotic GPI enzymes, the mutations G145C and C921A are likely to impair the enzyme function or stability. Case2:The propositus was a 8 yrs. old girl of Southern Italian origin with a history of chronic hemolytic anemia and occasional need for blood transfusion. At the age of 7 yrs. she was splenectomised reducing the need for blood transfusion. At the time of the study Hb was 9.4 g/dL, reticulocytes 364x109/L, total bilirubin 12.3 mg/dL. The propositus was homozygote for Gilbert’s syndrome. GPI activity was 20% of normal. The parents were not consanguineous, and displayed intermediate GPI activity (55% and 61% of normal). The sequence of complete erythrocyte GPI gene revealed the presence of two missense mutations, G301A (already described in an Italian patient by Baronciani et al Blood 88:2306,1996) and the new variant G1009A, which result in the aminoacidic substitutions Val101Met and Ala337Thr respectively.