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Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disease marked by chronic hemolytic anemia of various severity and frequent complications including gallstones, splenomegaly, iron overload, and others. Disease phenotype is highly heterogeneous and changes over time with children, adolescents and adult patients displaying different transfusion requirement and rates of complications. The diagnosis relies on the initial clinical suspicion in a patient with chronic hemolysis and exclusion of other more common congenital forms of hemolytic anemias; it is supported by the demonstration of reduced PK enzyme activity, and further confirmed by the detection of (homozygous or compound heterozygous) mutations of PKLR gene. Therapy is mainly supportive, with vitamin supplementation and transfusions (based on symptoms and patient growth rather than on fixed Hb thresholds). Splenectomy is widely performed, although it is less effective than in membrane defects and carries thrombotic and infectious risk. In the last decade, the allosteric PK enzyme activator mitapivat showed dramatic clinical benefit in clinical trials and gene therapy is also being studied to substitute the defective enzyme. In this review, we provide an insight in the current challenges of PKD diagnosis and management and discuss the future application of novel drugs and gene therapy, including a focus on quality of life.

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.

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.

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.

L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.

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.

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: 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.

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.

Two siblings of Italian origin with mild chronic haemolytic anaemia, psychomotor impairment and undetectable adenylate kinase (AK) activity are reported. The other red cell enzyme activities were normal except for a slight decrease of PFK. 2,3-DPG levels were increased in both siblings, and AMP decreased in one only. The parents were not consanguineous and displayed intermediate AK activity. The sequence of complete erythrocyte AK-1 cDNA showed the presence of a nonsense homozygous mutation at codon 107 (CGA --TGA, Arg --Stop) in the siblings. The mutation results in a truncated protein of 107 amino acids in comparison with the 194 of the normal one. Moreover a 37 bp deletion in the first part of exon 6 (from nt 326 to nt 362 of the cDNA sequence) was detected in one allele; this deletion is not likely to further affect the enzyme structure, being localized after the stop codon. The new variant was named AK Fidenza, from the origin of the patients.

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.

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

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.

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.