Evaluation of the main regulators of systemic iron homeostasis in pyruvate kinase deficiency.

Iron homeostasis and dyserythropoiesis are poorly investigated in pyruvate kinase deficiency (PKD), the most common glycolytic defect of erythrocytes. Herein, we studied the main regulators of iron balance and erythropoiesis, as soluble transferrin receptor (sTfR), hepcidin, erythroferrone (ERFE), and erythropoietin (EPO), in a cohort of 41 PKD patients, compared with 42 affected by congenital dyserythropoietic anemia type II (CDAII) and 50 with hereditary spherocytosis (HS). PKD patients showed intermediate values of hepcidin and ERFE between CDAII and HS, and clear negative correlations between log-transformed hepcidin and log-EPO (Person's r correlation coefficient = - 0.34), log-hepcidin and log-ERFE (r = - 0.47), and log-hepcidin and sTfR (r = - 0.44). sTfR was significantly higher in PKD; EPO levels were similar in PKD and CDAII, both higher than in HS. Finally, genotype-phenotype correlation in PKD showed that more severe patients, carrying non-missense/non-missense genotypes, had lower hepcidin and increased ERFE, EPO, and sTFR compared with the others (missense/missense and missense/non-missense), suggesting a higher rate of ineffective erythropoiesis. We herein investigated the main regulators of systemic iron homeostasis in the largest cohort of PKD patients described so far, opening new perspectives on the molecular basis and therapeutic approaches of this disease.

Case report: Transfusion independence and abolition of extravascular hemolysis in a PNH patient treated with pegcetacoplan.

More than half of patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with complement fraction C5 inhibitors experience residual anemia and hemolysis. This is partly due to the persistent activation of the complement cascade upstream C5, resulting in C3 deposition on PNH erythrocytes and extravascular hemolysis in the reticuloendothelial system. Pegcetacoplan is the first proximal C3 inhibitor to be approved for PNH basing on favorable efficacy and safety data in both naïve and eculizumab treated PNH. Here we report the first Italian patient treated with pegcetacoplan in a named patient program. The patient suffered from hemolytic PNH associated with CALR+ myeloproliferative neoplasm and was heavily transfusion dependent despite eculizumab therapy. Treatment with pegcetacoplan induced a dramatic improvement in Hb, along with normalization of unconjugated bilirubin and reticulocytes, as markers of extravascular hemolysis. Sequential laboratory workup showed the disappearance of C3 deposition on erythrocytes by direct anti-globulin test, the increase of PNH clone on erythrocytes, and a peculiar right shift of the ektacytometry curve. The drug was well tolerated, and the patient reported a significant improvement in his quality of life. Overall, pegcetacoplan appears a safe and effective option "ready to use" in the clinic for patients with PNH and suboptimal response to anti-C5 agents.

Pyruvate Kinase Deficiency: Current Challenges and Future Prospects.

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.

Effect of primary lesions in cytoskeleton proteins on red cell membrane stability in patients with hereditary spherocytosis.

We investigated by targeted next generation sequencing the genetic bases of hereditary spherocytosis in 25 patients and compared the molecular results with the biochemical lesion of RBC membrane obtained by SDS-PAGE analysis. The HS diagnosis was based on available guidelines for diagnosis of congenital hemolytic anemia, and patients were selected because of atypical clinical presentation or intra-family variability, or because presented discrepancies between laboratory investigation and biochemical findings. In all patients but 5 we identified pathogenic variants in SPTA1, SPTB, ANK1, SLC4A1, EPB42 genes able to justify the clinical phenotype. Interestingly, a correspondence between the biochemical lesion and the molecular defect was identified in only 11/25 cases, mostly with band 3 deficiency due to SLC4A1 mutations. Most of the mutations in SPTB and ANK1 gene didn't hesitate in abnormalities of RBC membrane protein; conversely, in two cases the molecular lesion didn't correspond to the biochemical defect, suggesting that a mutation in a specific cytoskeleton protein may result in a more complex RBC membrane damage or suffering. Finally, in two cases the HS diagnosis was maintained despite absence of both protein defect and molecular lesion, basing on clinical and family history, and on presence of clear laboratory markers of HS. The study revealed complex relationships between the primary molecular lesion and the final effect in the RBC membrane cytoskeleton, and further underlines the concept that there is not a unique approach to the diagnosis of HS.

Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

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.

Rasburicase-induced Methemoglobinemia: A Case Report and Literature Review.

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

Congenital Hemolytic Anemias: Is There a Role for the Immune System?

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 ß-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.

Clinical and Molecular Spectrum of Glucose-6-Phosphate Isomerase Deficiency. Report of 12 New Cases.

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.

'Gardos Channelopathy': a variant of hereditary Stomatocytosis with complex molecular regulation.

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.

Hereditary Xerocytosis due to Mutations in PIEZO1 Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families.

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. PIEZO1 gene 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.

A case of hereditary spherocytosis misdiagnosed as pyruvate kinase deficient hemolytic anemia.

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.

A new variant of phosphoglycerate kinase deficiency (p.I371K) with multiple tissue involvement: molecular and functional characterization.

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to MgADP, to form 3-phosphoglycerate and MgATP. Two isozymes encoded by distinct genes are present in humans: PGK-1, located on Xq-13.3, encodes a ubiquitous protein of 417 amino acids, whereas PGK-2 is testis-specific. PGK1 deficiency is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy; patients rarely exhibit all three clinical features. Nearly 40 cases have been reported, 27 of them characterized at DNA or protein level, and 20 different mutations were described. Here we report the first Italian case of PGK deficiency characterized at a molecular and biochemical level. The patient presented during infancy with hemolytic anemia, increased CPK values, and respiratory distress; the study of red blood cell enzymes showed a drastic reduction in PGK activity. In adulthood he displayed mild hemolytic anemia, mental retardation and severe myopathy. PGK-1 gene sequencing revealed the new missense mutation c.1112T>A (p.Ile371Lys). The mutation was not found among 100 normal alleles, and even if located in the third to the last nucleotide of exon 9, it did not alter mRNA splicing. The p.Ile371Lys mutation falls in a conserved region of the enzyme, near the nucleotide binding site. The mutant enzyme shows reduced catalytic rates toward both substrates (apparent k(cat) values, 12-fold lower than wild-type) and a decreased affinity toward MgATP (apparent K(m), 6-fold higher than wild-type). Moreover, it lost half of activity after nearly 9-min incubation at 45°C, a temperature that did not affect the wild-type enzyme (t(1/2)>1 h). The possible compensatory expression of PGK2 isoenzyme was investigated in the proband and in the heterozygote healthy sisters, and found to be absent. Therefore, the highly perturbed catalytic properties of the new variant p.Ile371Lys, combined with protein instability, account for the PGK deficiency found in the patient and correlate with the clinical expression of the disease.

CDAII presenting as hydrops foetalis: molecular characterization of two cases.

We report two patients with very severe congenital dyserythropoietic anemia presenting with hydrops foetalis, previously classified as "atypical" CDAs since they presented CDAII-like erythroblastic morphological features lacking other diagnostic CDAII markers. Molecular characterization of SEC23B gene, recently described as responsible of CDAII, revealed the presence of Glu109Lys/Arg701Cys and Glu109Lys/Cys66Tyr mutations, respectively. This finding leads to a re-classification of these cases and underlines phenotypic heterogeneity of CDAII, demonstrating for the first time that CDAII may be associated with hydrops foetalis and intrauterine death.

Congenital dyserythropoietic anemia type II (CDAII) is caused by mutations in the SEC23B gene.

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.

Clinical and hematologic features of 300 patients affected by hereditary spherocytosis grouped according to the type of the membrane protein defect.

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.

Recessive hereditary methemoglobinemia: two novel mutations in the NADH-cytochrome b5 reductase gene.

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.