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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.

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.

BACKGROUND: The present study aimed to use the targeted capture and sequencing technique to diagnose adult hereditary spherocytosis (HS). These results were compared with clinical features and laboratory examinations to explore the diagnosis of HS. METHODS: Whole blood and clinical data from ten patients with HS were collected. Genomic DNA was extracted, and a library was prepared. Exomes of patients with ten HS-related genes encoding red cell membrane skeleton protein were captured and sequenced. Bioinformatics analyses were carried out throughout the 1000 Genomes Project, ExAC, dbSNP147, and 1000 Normal Han Population databases. RESULTS: Gene mutations were found in 9 out of 10 cases of HS. Our data validation showed 90% specificity. Three types of gene mutations were found, including 6 cases of SPTB, 3 cases of ANK1, and 2 cases of SLC4A1. There were 4 mutation forms, including nonsense mutation, missense mutation, shear mutation, and code shift mutation, all of which were new, heterozygous mutations. These variations were predicted to be pathogenic in four databases. CONCLUSIONS: Our data demonstrate that targeted gene enrichment and sequencing methods were an efficient tool for determining genetic etiologies of red blood cell (RBC) membrane disorders and can facilitate accurate diagnosis and genetic counseling. They are also in good agreement with the clinical results.

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.

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.

A previously healthy 15-year-old girl was evaluated following five episodes of reddish urine discoloration after walking for approximately 30 min on a smooth roadway. In each episode, the discoloration lasted for four to five urinations and followed by normal urine dipstick tests. No other exercise-produced urine discoloration and no other symptoms were reported. Laboratory evaluation during the episodes revealed a reddish urine sample with 3+ hemoglobin/myoglobin and absence of hematuria. Full blood count, serum creatinine, liver function tests, and electrolyte levels were all within normal limits. Myoglobulinuria was excluded, since muscle enzymes were within normal limits. Blood smear analysis showed mild anisopoikilocytosis with stomatocytes and ovalocytes, leading to extended evaluation for erythrocyte disorders. This case is interesting in that the hemoglobinuria occurred after mild walking and was accompanied by erythrocyte morphological changes. This quiz discusses the differential diagnosis of hemoglobinuria with particular reference to the conditions of appearance (after walking) and emphasizes the importance of step-by-step investigations to reach a definitive diagnosis.

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 first case of a child with recessive hereditary methemoglobinemia type II with demonstrated cerebellar atrophy. This very rare blood disorder results in mild cyanosis, profound mental and motor impairment, and movement disorders in infancy and childhood. We suggest that children with unexplained severe encephalopathy and cerebellar atrophy should also be tested for hereditary methemoglobinemia type II.

BACKGROUND: Febrile nonhemolytic transfusion reactions occur in 0.12% of transfusions, usually during transfusion or within 4 to 6 hours after transfusion and are not medically dangerous. CASE REPORT: A patient with thalassemia from Togo with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile nonhemolytic transfusion reaction, is presented. Thirty-two hours after transfusion of 2 O D− red blood cell (RBC) units, the patient (phenotype A2 D+) developed fever and multiorgan failure and was admitted to the intensive care unit. Direct and indirect antiglobulin tests were negative on posttransfusion samples. Blood cultures and infectious diseases testing were negative. No malaria parasites were found at thick blood smear microscopic examination on Days 1 and 2 and the malaria rapid diagnostic test gave inconsistent results. Plasmodium total antibodies were detected in the serum at high levels. On Day 5, routine microscopic examination of blood smear revealed the presence of parasites in a very small number of RBCs. This finding was almost simultaneous to the availability of polymerase chain reaction testing results that were positive for P. falciparum. The sequential agglutination with anti-A antiserum allowed patient's and donors' RBCs to be separated and revealed that the parasitized cells were almost exclusively those of donors (14.4% vs. 0.029%). Malaria infection in implicated donors was excluded. CONCLUSION: In this patient with thalassemia with asymptomatic malaria, the infusion of two normal RBC units provided a favorable environment for a rapid parasite replication leading to a dramatic acute malaria attack.

Congenital dyserythropoietic anaemia type II (CDAII) is a rare autosomal recessive disease characterized by ineffective erythropoiesis, haemolysis, erythroblast morphological abnormalities, hypoglycosylation of some red blood cell membrane proteins, particularly band 3, and mutations in the SEC23B gene. We report the analysis of 101 patients from 91 families with a median follow-up of 23 years (range 0-65); 68 patients are newly reported. Clinical and haematological parameters were separately analysed in early infancy and thereafter, when feasible. Molecular analysis of the SEC23B gene confirmed the high heterogeneity of the defect, leading to the identification of 54 different mutations, 24 of which are newly described. To evaluate the genotype-phenotype correlation, patients were grouped according to their genotype (two missense mutations vs. one missense/one drastic mutation) and assigned to two different severity gradings based on laboratory data and on therapeutic needs; by this approach only a weak genotype-phenotype correlation was observed in the analysed groups.

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.

We reviewed clinical and molecular data of 23 consecutive unrelated patients affected by paroxysmal nocturnal hemoglobinuria (PNH) (19 with hemolytic PNH, 3 with aplastic anemia/PNH, and 1 with myelodysplasia/PNH syndrome) with a mean follow-up of 11.8 years. Five patients had thrombotic episodes, and 10 needed regular blood transfusions; 2 died for cerebral hemorrhage and kidney failure, and 2 spontaneously recovered from PNH. Twenty different PIG-A gene mutations were detected in 21/23 patients: 15 frameshift, 1 splicing, 2 nonsense, and 2 missense mutations. Two mutations (DelG341 and IVS2 +1g-a) were detected twice. A PIG-A mutated clone was also revealed in the two patients in complete clinical remission. One patient with aplastic anemia/PNH syndrome was treated with two courses of antilymphocyte globulin and cyclosporin with partial sustained response. Six patients were given rHu-EPO 150 U/kg/day s.c. for at least 6 months: one became transfusion-independent for 8 months and then discontinued treatment for clinical complications; one displayed a mean rise of Hb of 1.5 g/dL and is currently maintaining Hb levels higher than 9 g/dL after 54 months of therapy. Mutation specific quantitative-competitive PCR showed that the rise of hemoglobin was related to an increase of PIG-A negative molecules, suggesting that the efficacy of rHu-EPO therapy may be due to the stimulation of the abnormal clone.

Summary. Pyrimidine 5′-nucleotidase deficiency is a rare autosomal recessive disorder characterized by haemolytic anaemia, marked basophilic stippling and accumulation of pyrimidine nucleotides within the erythrocytes. The gene encoding for this enzyme (P5′N-1) has been cloned recently, and seven mutations have so far been identified in 11 unrelated families. We describe the haematological and molecular characteristics of six unrelated Italian patients affected by pyrimidine 5′-nucleotidase deficiency (one from northern and five from southern Italy). The sequence of the complete P5′N-1 gene showed the presence of four different new mutations: a missense mutation AAT–AGT at codon 190 (Asn–Ser), one splicing mutation (IVS9-1 g-c) and two frameshift mutations, DelG576 and InsGG743. Although the molecular defect was homozygous in all patients but one, parents' consanguinity could be confirmed in only one case. InsGG743 was detected in two cases, and DelG576 was found in three patients originating from southern Italy, suggesting a possible geographical distribution of the genetic defect. Haematological data showed the presence of peripheral spherocytosis in all cases, although only one had a concomitant membrane defect. An increase in serum ferritin levels was observed in the splenectomized patients, suggesting that the iron status of these subjects should be monitored and that they should be investigated for potential additional risk factors for iron accumulation.

ABSTRACT We evaluated the iron status and searched for mutations C282Y and H63D in the hereditary hemochromatosis gene ( HFE ) in 34 pyruvate kinase (PK)-deficient patients from 29 unrelated families. Nine had received multiple transfusions. Thirteen of the 25 nontransfused patients displayed increased serum ferritin concentration, in the absence of conditions known to raise this parameter. HFE genotype was abnormal in 9 of 34 patients. The allele frequency was 1.8% for mutation 845G→ (C282Y) and 16.1% for mutation 187C→:G (H63D). Nontransfused subjects with abnormal genotype had serum ferritin and transferrin saturation values significantly higher than those with wild-type genotype. Of the 12 adult nontransfused patients with increased iron status parameters, 1 was C282Y homozygous, 1 compound heterozygous for C282Y and H63D, 3 H63D heterozygous, and 7 had a normal HFE genotype. Serum ferritin and transferrin saturation were not related to hemoglobin, reticulocytes, and bilirubin concentration. At multivariate analysis serum ferritin was independently associated with age and gender, but not with splenectomy and HFE genotypes. The retrospective evaluation of the iron status profile of 10 patients (3 with abnormal and 7 with wild-type HFE genotype) with at least 10 years follow-up showed that overt iron accumulation requiring iron chelation had occurred only in the 3 patients (2 of whom were splenectomized) with the mutated HFE gene.

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(−2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(−2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3′ untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(−2)c lead to an alteration of the 5′ and 3′ splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

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.

Background: Congenital hemolytic anemias (CHAs) are a heterogeneous group of inherited RBC disorders including membrane and enzyme defects and dyserythropoietic anemias. Iron overload is a well recognized complication of hereditary hemoglobinopathies, both in transfusion-dependent and independent cases. However, little is known in congenital hemolytic anemias, with the exception of anecdotic reports in pyruvate kinase deficiency and dyserythropoietic anemias. Aim: to describe the clinical and hematological features at diagnosis and enrolment, to investigate iron overload by hepatic and cardiac T2* MRI, and to study inflammatory/regulatory cytokine profiles (IL-6, TNF-alpha, IFN-gamma, IL-10, IL-17) and hepcidin levels in patients with CHAs. Confounding factors such as hemocromatosis genotyping, metabolic syndrome, and hepatic viral profile were also considered. Methods: Between July 2015 and April 2016, 38 patients were enrolled (13 hereditary spherocytosis -HS, 3 hereditary stomatocytosis - HSt, 8 congenital dyserythropoietic anemia type II - CDAII, 13 pyruvate kinase deficiency - PKD, 1 glucose-phosphate isomerase deficiency). HS cases were enrolled on the basis of ferritin >300 ng/mL at diagnosis. Cytokine levels were detected in serum by ELISA. Comparisons were made by Students T test (continuous) and Fisher's exact test (categorical), and correlations by Pearson's linear coefficient. Results: The main clinical and hematological findings are shown in table. Median Hb values progressively decreased in the 4 groups considered, being close to normal in HS and moderately reduced in CDAII patients, whereas hemolytic parameters were comparable among groups. Consistently with clinical severity, ferritin values were particularly high in CDAII (together with transferrin saturation-TfS) and PKD patients, notwithstanding chelation in about half cases of both groups. Of note, only 2 PKD patients were transfusion-dependent, suggesting that other factors are involved in iron overload. Splenectomy had been performed in 17/38 (44.7%), mainly CDAII. Liver iron concentration (LIC) showed a great heterogeneity in all groups, with a trend towards higher values in CDAII; 16/36 (44%) patients had a LIC>4 mg/g DW (23%, 33%, 38% and 88% in HS, HSt, PKD and CDAII, respectively). Cardiac T2* value was normal in all subjects, with the exception of a HS and a CDAII case. Regarding possible cofactors, 12/16 displayed at least one of the following: 1 homozygous for HFE C282Y and 1 for H63D mutations, 3 HCV+, 4 BMI>25, 2 alcohol abuse, 3 heterozygous for HFE mutations. The following positive correlations were observed at enrolment: LIC and ferritin (r=0.68, p4), of whom 5 with the above listed cofactors. As regards cytokine levels, IL-10 was significantly increased in HS, PKD and CDAII groups compared with normal cases; TNF-alpha was decreased in HS and PKD, and IFN-gamma increased in HS and CDAII. Ferritin values were positively correlated with IL-6 and IFN-gamma, and TfS negatively with IL-6 (r= -0.38, p Conclusion: Iron overload is highly prevalent in CHAs, particularly in PKD and CDAII, is independent from transfusion support, and is also observed in cases with ferritin Table Table. Disclosures Barcellini: Agios: Consultancy.

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.

Background Hereditary spherocytosis is a very heterogeneous form of hemolytic anemia. The aim of this study was to relate the type of molecular defect with clinical and hematologic features and response to splenectomy using information from a large database of patients.Design and Methods Data from 300 consecutive patients with hereditary spherocytosis, grouped according to the results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis, were analyzed and the sensitivity of red cell osmotic fragility tests was compared in various subsets of patients.Results Band 3 and spectrin deficiencies were the most common protein abnormalities (54% and 31%, respectively); 11% of cases were not classified by the electrophoretic analysis. Spectrin deficiency was more frequently diagnosed in childhood and band 3 deficiency in adulthood. Hemoglobin concentration was slightly lower, spherocyte number and hemolysis markers higher in spectrin deficiency than in band 3 deficiency. The sensitivity of the osmotic fragility tests ranged from 48% to 95%, and was independent of the type and amount of the membrane defect. The association of the acidified glycerol lysis test and the NaCl test on incubated blood reached a sensitivity of 99%. Splenectomy corrected the anemia in patients with all subtypes of hereditary spherocytosis although spectrin-deficient patients still showed increased reticulocyte numbers and levels of unconjugated bilirubin. Splenectomy allowed the identification of the membrane defect in all the previously unclassified patients, most of whom had spectrin and/or ankyrin deficiency.Conclusions The definition of the red cell membrane defect in hereditary spherocytosis has no major clinical implications, but may be useful for a differential diagnosis from other hematologic disorders that mimic this hemolytic anemia.

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.

Introduction: Hereditary hemolytic anemias (HAs) is a group of heterogeneous disorders mainly due to defects of red cell (RBC) membrane and metabolism. HAs of RBC membrane are classified according to the morphological appearance on a blood smear: the more common are hereditary spherocytosis (HS), elliptocytosis, followed by stomatocytosis (dehydrated, DHSt and overhydrated, OHSt). Among RBC enzyme defects associated with chronic hemolysis, pyruvate kinase (PK) deficiency is the most common, followed by glucose-6-phosphate isomerase (GPI), pyrimidin 5' nucleotidase (P5'N), and more rarely, other enzymes deficiencies. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a battery of laboratory tests. Ektacytometry, and more recently, laser-assisted optical rotational cell analyser (LoRRca MaxSis, Mechatronics Instruments, NL), able to measure RBC deformability in osmotic gradient conditions (Osmoscan), is reported to be a useful tool in the detection of RBC membrane disorders and in particular for the differential diagnosis of HSt. Few data are available in other haemolytic anemias. Methods: We evaluated the diagnostic power of laser-assisted optical rotational cell analyser in 84 cases with RBC membrane disorders (69 HS, 7 HE, 7 DHSt, 1 OHSt) and 26 enzymopathies: 19 PK deficiency (14 not splenectomised), 4 GPI deficiency and 3 P5N deficiency. Moreover, we analysed 8 congenital diserythropoietic anemia type II (CDA II), of whom 3 not splenectomised. A reference curve interval was obtained from 89 healthy controls. The evaluated parameters were: Omin (osmotic value at which the EI reaches its minimum, coinciding with the 50% hemolysis in osmotic fragility assays), EImax (maximal deformability obtained near the isotonic osmolality), and Ohyper (osmotic value at which the EI reaches half of its maximum value, representing cellular hydration status). Results: Considering membrane defects, all the 69 HS, regardless the biochemical defect, showed typical altered ektacytometry curves, with a decreased EImax (0.516±0.05 vs 0.595±0.008 for controls, p Concerning RBC enzymopathies, we found that all the GPI deficient cases showed altered enlarged Osmoscan curves associated with significant increased Ohyper (539±36 vs 462±13.4, p We separately analysed splenectomised and not splenectomised patients. Interestingly, for some diseases (PK, P5N, CDAII and OHSt) Osmoscan curve after splenectomy, falls in a defined atypical area, regardless the pathology (Fig 1d). Conclusion: The Osmoscan curves were diagnostic for all the HS and HE cases analysed, with Omin and EImax parameters significantly different from controls. DHSt cases always showed typical left-shifted curve. Interestingly, Ohyper was firstly described increased in GPI deficiency, offering a new diagnostic tool for this rare enzyme defect. The interpretation of Osmoscan requires caution in splenectomised cases as splenectomy may interfere with the RBC deformability. In conclusion, the Osmoscan analysis performed by LoRRca Maxsis represents a useful and feasible first step screening test in the diagnosis of RBC membrane disorders and other rare haemolytic anemias. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

The PK-LR gene was studied in 23 patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase (PK) deficiency. Twenty-seven different mutations were detected among the 42 mutated alleles identified: 19 missense mutations, four splice site mutations and one nonsense, one single base deletion and two large deletions. Seventeen of them (107G, 278T, 403T, 409A, 661A, 859C, 958A, 1094T, 1190T, 1209A, 1232C, 1369G, 507A, IVS9 -1c, IVS9 +43c [corrected] del C224, del 5006bp IVS3--> nt 1431) were new. Although all the exons, the flanking regions and the promoter were sequenced in all cases, we failed to detect the second expected mutation in four subjects. To correlate genotype to phenotype, the molecular results were related to the biochemical properties of the mutant enzymes by an analysis of the three-dimensional structure of erythrocyte PK. The new mutant 409A, found in association with the large deletion of 5006 bp in a newborn baby who died soon after birth, was functionally characterized by mutagenesis and in vitro expression of the protein to investigate its contribution in the severity of the clinical pattern. However, the biochemical data obtained for the mutant enzyme cannot explain the severe anaemia found in the PK-deficient patient hemizygous for this mutation.

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS-PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in-frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt −7 to −3), and the other within exon 13 (nt 1494–1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences. At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478–499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.

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.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by an acquired PIG-A gene mutation. The clinical symptoms are intravascular hemolysis and hemoglobinuria. A variety of mutations in PIG-A gene have been so far reported. The aim of the study was to investigate the molecular defect in 8 patients with hemolytic PNH and in a 51 years old female who spontaneously recovered from severe hemolytic PNH (SL). The study of the entire codifying region and flanking intronic sequences of PIG-A gene was done by SSCP analysis and sequencing. 8 new and one known mutations were detected. Results are reported in the table. Mutation C55T has been already described in 5 PNH patients, but its nature was controversial. C55T was the only mutation found in patient SL. It was also detected in patient DTI, in association with T728C. To exclude the somatic origin of this variant, the mucous membrane cells were analyzed and found to be positive. C55T was also present in 1/100 normal alleles investigated, thus confirming its polymorphic nature. Mutations so far identified could be useful as molecular markers for monitoring the course of the disease during therapy.

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 4186 We report the case of a thalassemic patient with asymptomatic malaria in which the infection became clinically manifest only after blood transfusion, mimicking a febrile non haemolytic transfusion reaction. The patient was a 19 yr old thalassemic girl from Togo regularly and uneventfully transfused every 60-90 days since the age of 5 months and splenectomised at 13 yrs. Two malaria episodes in 1995 and 1997 were treated with quinine and halofantrin respectively. In February 2008 the transfusion interval shortened (15 days) and blood transfusions were constantly followed after 30-40 hours by high fever (39-4028C) and hypotension. The patient was referred to our Hospital to clarify and overcome the transfusion problems. Malaria attacks were excluded by the referring clinic. At admission, Hb was 5.9 g/dL, Hct 18.3%, MCV 71.8 fL, MCH 23.1 pg, MCHC 32.2%, reticulocytes 0.82%. WBC 13.1 × 109/L platelet count 953 × 109/L erythroblasts 12.7% of nucleated cells. Molecular analysis of β and α genes revealed double heterozygosity for β IVS1-1 G>A and β IVS2-849 A>G, associated with deletion 3.7 (-α3.7) at the heterozygous state. G6PD 6.29 IU/g Hb (ref.values 5.97±1), Pyruvate kinase 25.1 IU/gHb (ref. v. 11.9-16.1). Red cell osmotic fragility was decreased. The cytofluorimetric analysis of red cells labelled with eosin 5 maleimide gave normal results. Direct antiglobulin test was negative and red cell alloantibodies were not detected. The patient's blood group assessed by molecular biology testing was: genotype O1A2, phenotype A2; ccDee; K-k+; Jk(a+b-); Fy(a-b+); M+N+S+s-. The patient was given 2 crossmatch negative, filtered O neg units: the post-transfusion Hb values were 9.9 g/dL. Thirty-two hours later the patient developed fever (40°C) and hypotension followed by disseminated intravascular coagulation, metabolic acidosis and anuria. Direct and indirect antiglobulin tests were negative on a post-transfusion sample. Because of the rapid worsening of clinical conditions she was admitted to the Intensive Care Unit of our Hospital. Septic shock was excluded on the basis of negative blood cultures and infectious diseases testing. Microscopic examination of thick blood smear was negative for malaria parasites on 2 consecutive days and the malaria rapid diagnostic test (RDT) (Core Malaria Pan-PV-PF®, Core Diagnostics, U.K.-Effegiemme, Nerviano, Milano, Italy) gave inconsistent results. Anti Plasmodium total antibodies (Malaria EIA New Market Laboratories U.K.- Bio-Rad Italia) were detected in the serum at high levels (odd sample/cut off 22). Vital functions were supported by mechanical ventilation, amine administration and continuous venous-venous hemofiltration (CVVH). Broad spectrum antibiotic therapy was started. Fresh frozen plasma and two units of PRC were transfused on day 1 and 2 without increment of Hb level. Three days later, the examination of the peripheral blood smear for counting schistocytes revealed the presence of parasites in a very small number of red cells. This finding was almost simultaneous to the availability of PCR testing results that were positive for P. Falciparum. The differential agglutination with anti A antibody performed on a blood sample collected at admission to ICU allowed to separate patient's and donors' red cell. Counting the number of parasitized red cells in total blood and in the donor fraction blood revealed that the parasitized cells were almost exclusively those of donors (14.4 % vs 0.029%). Treatment with quinine chlorhydrate 500 mg i.v. every 8 hr for 3 days followed by oral quinine sulphate 500 mg every 8 hr for 3 days was successfully undertaken without significant side effects leading to dramatic improvement of clinical conditions and to eradication of P. falciparum. Two RBC units were effectively transfused without any reaction, and in the following two weeks the patient maintained stable haemoglobin values. After discharge, the transfusion interval returned to the previous values (60-90 days) without any post-transfusion reaction. We therefore think that the transfusion of normal, non malaria-resistant red cells fuelled the P. falciparum infection causing fever, DIC and acidosis giving rise to a very severe, atypical febrile non haemolytic transfusion related reaction. 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.

PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table: Pt Origin Hb (g/dL) Tx (n.) PK Activity (IU/gHb) Mutation Aminoacidic substitution SD Italy 15.1 0 10.6 107G / ? Ala36Gly/? TT Australia 8.9 0 nd 409A / del5006bp( IVS3-nt 1431) Ala137Thr / del ex 4-11 NR Italy 9 >50 5.5 661A /1209A Asp221Asn /Met403Ile NA Italy 9 >50 5.3 661A /1209A Asp221Asn /Met403Ile SA Italy 12 0 5.5 1456T/ 1209A Arg486Trp/ Met403Ile SC Italy nd nd 5.6 1529A/ 859C Arg510Gln/ Phe287Leu CM Italy 9.5 0 13.6 1456T/ 958A Arg486Trp/ Val320Met PS Italy 13.2 0 8 1094T /? Lys365Met /? VR Italy 10.5 0 10.7 1706A / ? Arg569Gln /? GR Guinea 14.4 0 7.6 1269A/ IVS9+43c Splice site/? Ref. Values 12.2–16 11.1–15.5 Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln. A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.