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At least 16 genetically determined conditions qualify as red blood cell enzymopathies. They range in frequency from ultrarare to rare, with the exception of glucose-6-phosphate dehydrogenase deficiency, which is very common. Nearly all these enzymopathies manifest as chronic hemolytic anemias, with an onset often in the neonatal period. The diagnosis can be quite easy, such as when a child presents with dark urine after eating fava beans, or it can be quite difficult, such as when an adult presents with mild anemia and gallstones. In general, 4 steps are recommended: (1) recognizing chronic hemolytic anemia; (2) excluding acquired causes; (3) excluding hemoglobinopathies and membranopathies; (4) pinpointing which red blood cell enzyme is deficient. Step 4 requires 1 or many enzyme assays; alternatively, DNA testing against an appropriate gene panel can combine steps 3 and 4. Most patients with a red blood cell enzymopathy can be managed by good supportive care, including blood transfusion, iron chelation when necessary, and splenectomy in selected cases; however, some patients have serious extraerythrocytic manifestations that are difficult to manage. In the absence of these, red blood cell enzymopathies are in principle amenable to hematopoietic stem cell transplantation and gene therapy/gene editing., (Copyright © 2021 by The American Society of Hematology.)

Hexokinase (EC 2.7.1.1, Adenosine Tri Phosphate (ATP): D-hexose-6-phosphotransferase) is a crucial regulatory enzyme of the glycolytic pathway (Embden-Meyerhof pathway). Hexokinase deficiency is associated with chronic non-spherocytic haemolytic anaemia (HA) with some exceptional cases showing psychomotor/mental retardation and fetus death. The proband is a four-and-half-year-old female child born of a four-degree consanguineous marriage hailing from South India with autosomal recessive congenital HA associated with developmental delay. She was well till 3 months of her age post an episode of diarrhoea when she was noted to be severely anaemic and requiring regular transfusions. The common causes of HA, haemoglobinopathies, red cell membranopathies and common red cell enzymopathies (G6PD, GPI, PK and P5N) were ruled out. Targeted analysis of whole exome sequencing (WES) using an insilico gene panel for hereditary anaemia was performed to identify pathogenic variants in the patient. Next-generation sequencing revealed a novel homozygous variant in hexokinase gene c.2714C>A (p. Thr905Lys) in exon-18. The pathogenic nature of the variant p. Thr905Lys in the HK1 gene was confirmed collectively by biochemical and molecular studies. Insilico analysis (PolyPhen-2, Provean, Mutation Taster) predicted the variant to be severe disease causing. Multiple sequence alignment demonstrated the conservation of p. Thr905 across the species. The impact of the mutation on the protein structure was studied by PyMOL and Swiss Protein databank viewer., Competing Interests: Competing interests: Authors BS and SJ are employed by the company Mediscan Systems, No. 197, Dr. Natesan Road, Mylapore, Chennai-600004., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13) is a metalloprotease responsible for cleavage of ultra-large von Willebrand factor (VWF) multimers. Severely deficient activity of the protease can trigger an acute episode of thrombotic thrombocytopenic purpura (TTP). Our understanding of the pathophysiology of TTP has allowed us to grasp the important role of ADAMTS13 in other thrombotic microangiopathies (TMAs) and thrombotic disorders, such as ischemic stroke and coronary artery disease. Through its action on VWF, ADAMTS13 can have prothrombotic and proinflammatory properties, not only when its activity is severely deficient, but also when it is only moderately low. Here, we will discuss the biology of ADAMTS13 and the different assays developed to evaluate its function in the context of TTP, in the acute setting and during follow-up. We will also discuss the latest evidence regarding the role of ADAMTS13 in other TMAs, stroke, and cardiovascular disease. This information will be useful for clinicians not only when evaluating patients who present with microangiopathic hemolytic anemia and thrombocytopenia, but also when making clinical decisions regarding the follow-up of patients with TTP., (© 2018 by The American Society of Hematology.)

Background: Genetic variants of inosine triphosphatase (ITPA) that confer reduced ITPase activity are associated with protection against ribavirin(RBV)-induced hemolytic anemia in peginterferon(IFN)/RBV-based treatment of hepatitis C virus (HCV). Patients with reduced ITPase activity showed improved treatment efficacy when treated with IFN/RBV. In addition, a genetic polymorphism near the IL28B gene is associated with an improved response to IFN/RBV treatment. RBV has been an important component of IFN-containing regimens, and is currently recommended in combination with several IFN-free regimens for treatment of harder to cure HCV infections., Aim: To evaluate whether genetic variations that reduce ITPase activity impact RBV-induced anemia in IFN-free/RBV regimens., Methods: In this study, genetic analyses were conducted in the PEARL-IV trial to investigate the effect of activity-reducing ITPA variants as well as IL28B polymorphism on anemia, platelet (PLT) counts, and virologic response in HCV genotype1a-infected patients treated with the direct-acting antiviral (DAA) regimen of ombitasvir/paritaprevir/ritonavir and dasabuvir±RBV., Results: Reduction in ITPase activity and homozygosity for the IL28Brs12979860 CC genotype protected against RBV-induced anemia. In patients receiving RBV, reduced ITPase activity was associated with reduced plasma RBV concentration and higher PLT counts. ITPase activity had no impact on response to DAA treatment, viral kinetics, or baseline IP-10 levels., Conclusions: Our study demonstrates that genetics of ITPA and IL28B may help identify patients protected from RBV-induced anemia when treated with IFN-free regimens. Our work demonstrates for the first time that IL28B genetics may also have an impact on RBV-induced anemia. This may be of particular significance in patients with difficult-to-cure HCV infections, such as patients with decompensated cirrhosis where RBV-containing regimens likely will continue to be recommended., Competing Interests: AV, JWD, MA, JS, JZ, EOD, DEC, and JW are AbbVie employees and may hold AbbVie stock/options. ML is an employee of the University of Gothenberg, Sweden and has no further relationships to disclose. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

It has become easier to acquire patients' clinical information from clinical laboratories using the electronic medical record system. However, it is possible that clinical laboratories do not consider abnormal laboratory data when elucidating the pathological mechanism of diseases in patients when their clinical information is obtained from the electronic medical records. In this reversed clinicopathological conference, we analyzed the laboratory data of a patient with elevated lactate dehydrogenase (LD) levels and anemia. Although the presence of hemolysis was clear from the elevated LD levels, anemia, and decreased haptoglobin levels, the ,excessive increase in the LD level suggested a particular mechanism of hemolysis. Drug intake was sus- pected on the basis of a prolonged prothrombin time with a normal activated partial thromboplastin time. At the conference, these findings led to in-depth discussions. We elucidated the pathological mechanism in this patient based only on the laboratory data, with intentionally restricted clinical information, and again recog- nized the importance of analyzing laboratory data thoroughly. Training modules for medical technologists and doctors in clinical laboratories that provide information regarding elucidating pathological mechanisms using laboratory data may prove useful for improving the nature of comments in laboratory data reports. In addition, clinicians should also receive training to elucidate the pathological mechanism of a disease on the basis of only laboratory data, as well as training for diagnosing patients on the basis of clinical information and physical examination findings. [Review].

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phosphoglycerate kinase (PGK) deficiency, a rare X-linked inherited disorder, manifests as various combinations of hemolytic anemia, neurological dysfunction, and myopathy. We report a Japanese boy with PGK deficiency presenting as chronic hemolytic anemia. The diagnosis of PGK1 deficiency was made at 11 months of age on the basis of low PGK enzyme activity (36.7 IU/g Hb; normal, 264-326 IU/g Hb) and the identification through PGK1 gene sequencing of a novel missense mutation: c. 1180A>G at exon 10. The mutation, which has been designated PGK-Aoto, results in a Thr394Ala amino-acid substitution at β-strand L. Because β-strand L plays an important role in the function of the hinge connecting the two domains of PGK, the Thr394Ala substitution may perturb this motion. At 3 years of age the patient has transfusion-dependent hemolytic anemia but no evidence of neuromuscular disease or developmental delay. Long-term follow-up will be needed to identify possible future clinical manifestations.

That primaquine and other drugs can trigger acute haemolytic anaemia in subjects who have an inherited mutation of the glucose 6-phosphate dehydrogenase (G6PD) gene has been known for over half a century: however, these events still occur, because when giving the drug either the G6PD status of a person is not known, or the risk of this potentially life-threatening complication is under-estimated. Here we review briefly the genetic basis of G6PD deficiency, and then the pathophysiology and the clinical features of drug-induced haemolysis; we also update the list of potentially haemolytic drugs (which includes rasburicase). It is now clear that it is not good practice to give one of these drugs before testing a person for his/her G6PD status, especially in populations in whom G6PD deficiency is common. We discuss therefore how G6PD testing can be done reconciling safety with cost; this is once again becoming of public health importance, as more countries are moving along the pathway of malaria elimination, that might require mass administration of primaquine. Finally, we sketch the triangular relationship between malaria, antimalarials such as primaquine, and G6PD deficiency: which is to some extent protective against malaria, but also a genetically determined hazard when taking primaquine., (© 2013 John Wiley & Sons Ltd.)

In areas of low malaria transmission, it is currently recommended that a single dose of primaquine (0.75 mg base/kg; 45 mg adult dose) be added to artemisinin combination treatment (ACT) in acute falciparum malaria to block malaria transmission. Review of studies of transmission-blocking activity based on the infectivity of patients or volunteers to anopheline mosquitoes, and of haemolytic toxicity in glucose 6-dehydrogenase (G6PD) deficient subjects, suggests that a lower primaquine dose (0.25 mg base/kg) would be safer and equally effective. This lower dose could be deployed together with ACTs without G6PD testing wherever use of a specific gametocytocide is indicated.

AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(-/-)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(-/-,-/-)] mice by mating A3(-/-) mice with CBA-Pk-1slc/Pk-1slc mice [PK(-/-)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(-/-,-/-) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(-/-) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(-/-) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(-/-) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction., (© 2012 The Authors Genes to Cells © 2012 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

The pathophysiology of oxidative hemolytic anemia is closely associated with hemoglobin (Hb) stability; however, the mechanism of how Hb maintains its stability under oxidative stress conditions of red blood cells (RBCs) carrying high levels of oxygen is unknown. Here, we investigated the potential role of peroxiredoxin II (Prx II) in preventing Hb aggregation induced by reactive oxygen species (ROS) using Prx II knockout mice and RBCs of patients with hemolytic anemia. Upon oxidative stress, ROS and Heinz body formation were significantly increased in Prx II knockout RBCs compared to wild-type (WT), which ultimately accelerated the accumulation of hemosiderin and heme-oxygenase 1 in the Prx II knock-out livers. In addition, ROS-dependent Hb aggregation was significantly increased in Prx II knockout RBCs. Interestingly, Prx II interacted with Hb in mouse RBCs, and their interaction, in particular, was severely impaired in RBCs of patients with thalassemia (THAL) and sickle cell anemia (SCA). Hb was bound to the decameric structure of Prx II, by which Hb was protected from oxidative stress. These findings suggest that Prx II plays an important role in preventing hemolytic anemia from oxidative stress by binding to Hb as a decameric structure to stabilize it., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs., Objectives: To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in as mall and selected group of Beagles and West Highland White Terriers (WHWT)., Animals: Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other),WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1)., Methods: Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia., Results: A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37)., Conclusions and Clinical Importance: Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency.

Erythrocyte enzyme deficiencies do not usually shorten life expectancy except for PK deficiency in dogs and the potential for PFK-deficient dogs to die during hemolytic crises. In addition, erythrocyte enzyme deficiencies are uncommon or rare, so they are generally not seriously considered in the differential diagnosis of anemia until common causes of anemia have been excluded. However, unique clinical and/or laboratory findings like sporadic hemoglobinuria in English Springer spaniels (PFK deficiency) may quickly point to the possibility of an inherited erythrocyte enzyme defect. The ability to diagnose deficient or carrier animals allows for the possibility of eliminating these undesirable traits in future breeding. Continued research is needed to document additional enzyme deficiencies that likely occur and to develop additional DNA-based assays that are especially important in the recognition of heterozygous or carrier animals that have no clinical signs.

Erythrocyte enzymes participate in the main interactions promoting utilization of glucose-glycolytic, pentosophosphate cycles and glutation system. In this report we study on erythrocyte G6PD deficiency which is the impairment related to the gender and expressed with development of acute drug-associated hemolytic anemia. Out of 13187 studied subjects 122 showed carrying of deficiency of erythrocyte G6PD activity, from them 98 (80.3%) subjects were male, and 24 (19.7%) female. As a whole, among the revealed in the population studies, and also verified in clinic of the persons with deficiency of erythrocyte G6PD there were marked different pathological phenotypes: hereditary nonspherecytary hemolytic anemia, acute drug-induced hemolytic anemia, asymptomatic gene carrying and, selected by us disease with few symptoms. As a whole, among the revealed in the population studies, and also verified in clinic of the persons with deficiency of erythrocyte G6PD there were marked different pathological phenotypes: hereditary nonspherecytary hemolytic anemia, acute drug-induced hemolytic anemia, asymptomatic gene carrying and, selected by us disease with few symptoms.

Pyruvate kinase deficiency is a chronic illness with age specific consequences. Newborns suffer life-threatening hemolytic crisis and hyperbilirubinemia. Adults are at risk for infections because of asplenia, pregnancy-related morbidity, and may suffer organ damage because of systemic iron overload. We describe 27 Old Order Amish patients (ages 8 months-52 years) homozygous for c.1436G>A mutations in PKLR. Each subject had a predictable neonatal course requiring packed red blood cell transfusions (30 ± 5 mL/kg) to control hemolytic disease and intensive phototherapy to prevent kernicterus. Hemochromatosis affected 29% (n = 4) of adult patients, who had inappropriately normal serum hepcidin (34.5 ± 12.7 ng/mL) and GDF-15 (595 ± 335pg/mL) relative to hyperferritinemia (769 ± 595 mg/dL). A high prevalence of HFE gene mutations exists in this population and may contribute to iron-related morbidity. Based on our observations, we present a strategy for long-term management of pyruvate kinase deficiency., (2011 Wiley-Liss, Inc.)

G6PD(Mahidol) enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD(Mahidol) enzyme was characterized by molecular modeling to understand its kinetics. G6PD(Mahidol), G6PD(G487A) and G6PD(WT) proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD(WT), the Km, and Vmax of NADP+ with G6PD(G487A) were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD(G487A) showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD(G487A) was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD(G487A) alpha-helix d and beta-strand E, besides the conformation of beta-strand D. In conclusion, the biochemical and structural properties of G6PD(G487A) and G6PD(WT) enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

Over the last decade, bortezomib moved in a stepwise fashion from a benchside promise into a bedside reality and is currently an important tool in the treatment of plasma cell disorders. This review focuses on the relationship between bortezomib and hemolytic anemia. In animal models with lupus-like disease, this agent was shown to deplete the auto-reactive plasma cells and serum autoantibody levels. In humans, two isolated reports advocate the efficacy of bortezomib in autoimmune hemolytic anemia, but important concerns remain with data interpretation and length bias. Conversely, bortezomib may be causative of hemolytic anemia, as reported in a cohort of patients with chronic lymphocytic leukemia [corrected]. Concerted efforts of both basic and clinical researchers are necessary to further explore the safety and efficacy of this agent in nonmalignant disorders, including autoimmune disorders and anemias.

Introduction: Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare recessive disorder of cholesterol metabolism characterized by the absence of high density lipoprotein (HDL) and the triad of corneal opacification, hemolytic anemia and glomerulopathy., Patients: We here report on FLD in three siblings of a kindred of Moroccan descent with HDL deficiency. In all cases (17, 12 and 3 years of age) corneal opacification and proteinuria were observed. In the 17-year-old female proband, anemia with target cells was observed., Results: Homozygosity for a mutation in LCAT resulted in the exchange of cysteine to tyrosine at position 337, disrupting the second disulfide bond in LCAT. LCAT protein and activity were undetectable in the patients' plasma and in media of COS7 cells transfected with an expression vector with mutant LCAT cDNA. Upon treatment with an ACE inhibitor and a thiazide diuretic, proteinuria in the proband decreased from 6g to 2g/24h., Conclusion: This is the first report that FLD can cause nephropathy at a very early age., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked disorder, is usually observed in hemizygote males and very rarely in females. The G6PD class 1 variants, very uncommon, are associated with chronic hemolytic anemia. Here we report a Portuguese woman who suffered in her sixties from a chronic hemolytic anemia due to G6PD deficiency. Molecular studies revealed heterozygosity for an in-frame 18-bp deletion, mapping to exon 10 leading to a deletion of 6 residues, 362-367 (LNERKA), which is a novel G6PD class 1 variant, G6PD Tondela. Two of her three daughters, asymptomatic, with G6PD activity within the normal range, are heterozygous for the same deletion. The patient's leukocyte and reticulocyte mRNA studies revealed an almost exclusive expression of the mutant allele, explaining the chronic hemolytic anemia. Patient whole blood genomic DNA HUMARA assay showed a balanced pattern of X chromosome inactivation (XCI), but granulocyte DNA showed extensive skewing, harboring the mutated allele, implying that in whole blood, lymphocyte DNA, with a very long lifetime, may cover up the current high XCI skewing. This observation indicates that HUMARA assay in women should be assessed in granulocytes and not in total leukocytes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer conserved throughout evolution and important for energy sensing in all eukaryote cells. AMPK controls metabolism and various cellular events in response to both hormones and changes in cellular energy status. The γ subunit senses intracellular energy status through the competitive binding of AMP and ATP. We show here that targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing the sequestration of abnormal erythrocytes. Prkag1(-/-) mice displayed splenomegaly and iron accumulation due to compensatory splenic erythropoiesis and erythrophagocytosis. Moreover, AMPKγ1-deficient erythrocytes were highly resistant to osmotic hemolysis and poorly deformable in response to increasing shear stress, consistent with greater membrane rigidity. No change in cytoskeletal protein composition was observed; however, the phosphorylation level of adducin, a protein promoting the binding of spectrin to actin, was higher in AMPKγ1-deficient erythrocytes. Together, these results demonstrate that AMPKγ1 subunit is required for the maintenance of erythrocyte membrane elasticity.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common form of red blood cell enzymopathy. The disorder has reached polymorphic frequencies in different parts of the world due to the relative protection conferred against malaria. G6PD is a housekeeping X-linked gene encoding the first enzyme of the pentose phosphate pathway, an NADPH-producing dehydrogenase. Because erythrocytes do not generate NADPH in any other way than pentose phosphate pathway, they are more susceptible than any other cells to oxidative damages. G6PD deficiency is a prime example of a hemolytic anemia due to an interaction between an intracorpuscular cause and an extracorpuscular cause, because in the majority of cases an exogenous agent triggers hemolysis. Hemolysis, in fact, can be caused by exposure to oxidant agents. Although studies performed on epidemiology, genetics and molecular biology have broaden the information on G6pd deficiency, there are still no reliable and validated methods to test drug hemolytic potential in G6PD deficient patients. The review gives an overview of current knowledge on G6pd deficiency and on the methods that have been developed so far in order to identify drugs causing acute hemolytic anemia in G6pd deficiency. Moreover, we discuss the new potential preclinical strategies to assess, in vitro and in vivo, drug hemolytic risks.

Glycolysis is one of the principle pathways of ATP generation in cells and is present in all cell tissues; in erythrocytes, glycolysis is the only pathway for ATP synthesis since mature red cells lack the internal structures necessary to produce the energy vital for life. Red cell deficiencies have been detected in all erythrocyte glycolytic pathways, although their frequencies differ owing to diverse causes, such as the affected enzyme and severity of clinical manifestations. The number of enzyme deficiencies known is endless. The most frequent glycolysis abnormality is pyruvate kinase deficiency, since around 500 cases are known, the first of which was reported in 1961. However, only approximately 200 cases were due to mutations. In contrast, only one case of phosphoglycerate mutase BB type mutation, described in 2003, has been detected. Most mutations are located in the coding sequences of genes, while others, missense, deletions, insertions, splice defects, premature stop codons and promoter mutations, are also frequent. Understanding of the crystal structure of enzymes permits molecular modelling studies which, in turn, reveal how mutations can affect enzyme structure and function.

Glucose-6-phosphate isomerase (GPI), a homodimeric enzyme, catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate. In mammals, it can also act as an autocrine motility factor, neuroleukin, and maturation factor. Deficiency of the enzymatic activity in red blood cells causes nonspherocytic hemolytic anemia in human. To gain a more complete understanding of the molecular basis for the hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants were expressed in Escherichia coli and functionally characterized. Conclusions are as follows: (1) mutations usually have negative influences on catalytic parameters, particularly k(cat), as well as structure stability; (2) mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k(cat), despite lesser extents; (3) mutations decrease the enzyme tolerance to heat or SDS by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T); (4) A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an important cause of hemolytic anemia worldwide. Severely affected patients have chronic hemolysis with exacerbations following oxidative stress. Mutations causing severe chronic non-spherocytic hemolytic anemia (CNSHA) commonly cluster in Exon 10, a region important for protein dimerization. An African-American male presented at age 2 weeks with pallor and jaundice, and was found to have hemolytic anemia with G6PD deficiency. His severe clinical course was inconsistent with the expected G6PD A(-) variant. DNA sequencing revealed two common mutations (A(-)) and a third novel Exon 10 mutation. This inherited haplotype represents a novel triple G6PD coding mutation causing chronic hemolysis.

The oxidative status of cells is determined by the balance between pro-oxidants and antioxidants. Pro-oxidants, referred to as reactive oxygen species (ROS), are classified into radicals and nonradicals. The radicals are highly reactive due to their tendency to accept or donate an electron and attain stability. When cells experience oxidative stress, ROS, which are generated in excess, may oxidize proteins, lipids and DNA - leading to cell death and organ damage. Oxidative stress is believed to aggravate the symptoms of many diseases, including hemolytic anemias. Oxidative stress was found in the beta-hemoglobinopathies (sickle cell anemia and thalassemia), glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, congenital dyserythropoietic anaemias and Paroxysmal Nocturnal Hemoglobinuria. Although oxidative stress is not the primary etiology of these diseases, oxidative damage to their erythroid cells plays a crucial role in hemolysis due to ineffective erythropoiesis in the bone marrow and short survival of red blood cells (RBC) in the circulation. Moreover, platelets and polymorphonuclear (PMN) white cells are also exposed to oxidative stress. As a result some patients develop thromboembolic phenomena and recurrent bacterial infections in addition to the chronic anemia. In this review we describe the role of oxidative stress and the potential therapeutic potential of anti-oxidants in various hemolytic anemias.

We present the results of screening for pyruvate kinase (PK) deficiency on a cohort of 146 patients pre-selected from 4017 individuals by hematological index analysis. On average the PK activity levels measured in this cohort study were about 1.9% IU/g Hb while the activity measured in 85 healthy adults with normal erythrocyte indexes was in the range of 3.9-9.8 IU/g Hb. We were able to define 14 different mutations in the coding sequence of the R-PK gene in 74 individuals with low enzyme activity. The most common were the G1168A and G1529A mutations at exon 11 occurring in 54% of the cases. Other mutations occurring more than once were C1492T, C1456T, G1291A, C1594T, G787A, G994A, and G1010C. The polymorphism at nt 1705 was in linkage disequilibrium with the A and C polymorphism, which indicated a multi-centric origin of the mutation. Further study of the promoter region and intron/exon boundary is under investigation.

Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, being present in more than 400 million people worldwide. The global distribution of this disorder is remarkably similar to that of malaria, lending support to the so-called malaria protection hypothesis. G6PD deficiency is an X-linked, hereditary genetic defect due to mutations in the G6PD gene, which cause functional variants with many biochemical and clinical phenotypes. About 140 mutations have been described: most are single base changes, leading to aminoacid substitutions. The most frequent clinical manifestations of G6PD deficiency are neonatal jaundice, and acute haemolytic anaemia, which is usually triggered by an exogenous agent. Some G6PD variants cause chronic haemolysis, leading to congenital non-spherocytic haemolytic anaemia. The most effective management of G6PD deficiency is to prevent haemolysis by avoiding oxidative stress. Screening programmes for the disorder are undertaken, depending on the prevalence of G6PD deficiency in a particular community.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemoglobin S (HbS) are very common genetic disorders in sub Saharan Africa, where malaria is endemic. These genetic disorders have been associated with protection against malaria and are therefore under strong selection pressure by the disease. In November-December 2003, we conducted a cross-sectional survey to determine the prevalence of G6PD deficiency and HbS in the population and relate these to malaria infection and haemoglobin levels in lowland and highland areas of differing malaria transmission patterns of Muheza, Tanzania. Blood samples from 1959 individuals aged 6 months to 45 years were collected. A total of 415 (21%) and 1181 (60%) samples were analysed for G6PD deficiency and HbS, respectively. Malarial parasite prevalence was 17.2% (114/1959) in the highlands and 39.6% (49/1959) in the lowlands. Lowlands had higher prevalence of G6PD deficiency and HbS than highlands (G6PD deficiency = 11.32% (24/212) versus 4.43% (9/203), P = 0.01, and HbS = 16.04% (98/611) versus 6.32% (36/570), P = 0.0001). Logistic regression model showed an association between G6PD deficiency and altitude [lowlands] (Odds ratio [OR] 3.4, 95% CI = 1.49; 7.90, P = 0.004). In the lowlands, G6PD deficient individuals had lower mean haemoglobin (10.9g/dl) than normal ones (12.8g/dl), P = 0.01. These findings show that high malaria transmission in the lowlands might have selected for G6PD deficiency and HbS.

We report two previously undescribed mutations of the phosphoglycerate kinase gene (PGK1) leading to enzyme deficiency. In both cases, the patients were of Spanish origin and they exhibited a severe life-long chronic haemolytic anaemia associated with progressive neurological impairment. Sequence analysis of the first patient's entire PGK1 gene found a novel missense mutation (140T > A). This mutation caused an amino acid change of Ile to Asn at 46th position from the NH(2)-terminal serine residue (Ile46Asn), which has been called PGK-Barcelona based on the place of origin of the patient. In the second patient, a G to A transversion was discovered at nucleotide 958 (958G > A). This caused a Ser319Asn amino acid substitution. Since this mutation had not been previously described, the provisional name of PGK-Murcia was given to this deficient enzyme. The crystal structure of porcine PGK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. In both cases, the mutations did not modify any of the PGK binding sites for ATP or 3PG, so their consequence is related to a loss of enzyme stability rather than a decrease of enzyme catalytic function.

We report a rare case of severe hemolytic anemia accompanied by moderate renal insufficiency after mitral valve repair. Although the degree of the residual mitral regurgitation was less than 1+ during the first three weeks after the operation, the maximum lactate dehydrogenase (LDH) was up to 7,430 U/l and the minimum hemoglobin was 4.9 g/dl. The mitral valve replacement successfully resolved the hemolysis, but the renal function did not completely recover.

The hemolytic anemia development caused by phenylhydrazine injection (7 mg/100 g b.w.) was shown to be caused by the decreasing of both catalase activity and glutathione content in erythrocytes, and by the increasing of spontaneouse hemolysis level of these cells in blood stream. The increasing of heme oxygenase activity and TBA-active products in rat heart and vessels were revealed 24 hrs after phenylhydrazine injection. Possible mechanisms of heme oxygenase-1 induction under hypoxia as response to the hemolytic anemia development and it's role in defense of the cells from damage are discussed.

Mutations leading to red cell enzyme deficiencies can be associated with diverse phenotypes that range from hemolytic anemia, methemoglobinemia, polycythemia, and neurological and developmental abnormalities. While most of these mutations occur sporadically, some such as common glucose-6-phosphate dehydrogenase (G6PD) mutants are endemic and rarely cause disease. Common G6PD mutants likely reached their prevalence because they provide some protection against severe malarial complications. In this review G6PD, pyruvate kinase, 5' nucleotidase, and cytochrome b5 reductase deficiencies will be discussed in greater detail. Limitations of commonly used screening tests for detection of these disorders will also be emphasized, as well as emerging knowledge about non-enzymatic function of the glycolytic enzymes.

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.

The type-I isoform of pyrimidine 5'-nucleotidase (P5N-I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N-I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87-bp sequence. The sequence is located 6.2-kb downstream of the exon 2 and 2.7-kb upstream of the exon 3 sequence; consequently, the P5N-I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N-I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients were homozygous for each mutation. The mutant P5N-I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos-7. The degradation of P5N with 425C (L142P) was significantly faster than a wild-type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin-proteasome pathway.

Volatile alkyl nitrites have been used during the past decades for "recreational purposes," and for intensifying sexual experience. Their use has been associated with methemoglobinemia and hemolysis. We report three patients who presented over the past year with acute hemolysis after inhalation of butyl nitrite, two of them had glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Severe deficiency of von Willebrand factor (VWF)-cleaving protease (ADAMTS-13) activity (<5% of normal) is a specific finding for acute idiopathic thrombotic thrombocytopenic purpura (TTP), a disorder that presents as thrombocytopenia, microangiopathic hemolytic anemia, and often organ dysfunction such as neurological disturbances or renal failure, and fever. Between January 2001 and July 2003, ADAMTS-13 activity was determined in plasma samples of 396 consecutive patients referred to our laboratory for diagnostic purposes. Plasma samples with ADAMTS-13 activity less than 5% were in addition tested for the presence of inhibitory antibodies. Patients were assigned to 10 predefined clinical categories according to information provided by the referring clinician: thrombotic microangiopathy (TMA) not further specified; neoplasia- or chemotherapy-associated TMA; TMA following hematopoietic stem cell transplantation; TMA with additional/alternative disorder; idiopathic TTP; hemolytic-uremic syndrome (HUS) not specified; HUS with diarrhea prodrome (D+HUS); atypical HUS; other hematological disorder; and no clinical information available. Severe ADAMTS-13 deficiency was found in 69 (17%) patients, including 42 with acquired idiopathic TTP, either at initial presentation or at relapse, 14 with confirmed or suspected hereditary TTP, 10 with TMA not further specified, two with neoplasia- or chemotherapy-associated TMA, and one in continued clinical remission 3.4 years after splenectomy for plasma-refractory TTP. Forty-three (62%) patients with ADAMTS-13 activity less than 5% displayed inhibitory antibodies. Severe ADAMTS-13 deficiency was found in 60% of patients diagnosed with acute idiopathic TTP, but in none of 130 patients diagnosed with HUS or in any of the 14 patients with hematopoietic stem cell transplantation-associated TMA. Thus, plasma ADAMTS-13 activity less than 5% does not identify all patients clinically diagnosed with TTP, and severe ADAMTS-13 deficiency is not invariably associated with clinical manifestations of microvascular platelet clumping.

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine -248 and 2 single nucleotide substitutions, nucleotide (nt) -324T>A and nt -83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream -324T>A and -248delT mutations were nonfunctional polymorphisms. In contrast, the -83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt -87 and nt -83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

A feature of a previously proposed mechanism of erythrocyte shape control is a control of the shape by the Donnan equilibrium ratio. An enzymopathy of the glycolytic pathway and nucleotide metabolism could alter the Donnan ratio, since it alters levels of phosphate intermediates and of end-products carbonic gas (CO(2)) and ammonia gas (NH(3)) which are ionized at the erythrocyte pH. Thus, a hypothesis of haemolysis in haemolytic anaemias associated with enzymopathies of the glycolytic pathway and nucleotide metabolism would be that it results from an inhibition of the mechanism by a modification of the Donnan ratio. This hypothesis seems plausible as it would explain coherently observations in these haemolytic anaemias.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hemolytic enzymopathy affecting 3% of Southern Chinese males. Among 275 adult allogeneic hematopoietic stem cell transplantations (SCT), five cases (1.8%) each of donors and recipients were G6PD deficient. Among 107 autologous SCT, four patients (3.7%) were G6PD deficient. All subjects were male, except for two female patients with chronic myeloid leukemia (CML). The incidence of G6PD deficiency in female CML patients was significantly higher than the background female incidence (P = 0.004), but comparable with that in the males (P = 0.664). There was no significant hemolysis or delay in red cell engraftment, and all but one patient converted to donor G6PD screening status. One female patient achieved partial correction of her G6PD status and relapsed at 10 months. We suggest that G6PD deficiency should be tested for in all marrow donors and recipients in susceptible populations. From our data, there is a suggestion of increased clinical incidence of G6PD deficiency in female patients with multi-lineage clonal marrow disorders.

Red blood cells (RBCs) express two hexokinase (HK) isoforms, HK-I and HK-R. Both isozymes are generated from the HK-I gene by use of an alternate promoter. Gene structure and exon-intron organization of the HK-I gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. HK-R has been shown as an erythroid-specific isozyme whose expression is turned on in the early erythroid-progenitors and is significantly induced during their differentiation. HK-R unfolds major HK activity in immature RBCs and is rapidly degraded during the maturation process. HK-I has a porin-binding domain in its N-terminus. Recent studies have shown that HK isozymes with a porin-binding domain play a role in mitochondrial integrity, suggesting that HK-I-deficient erythroid cells might be eliminated by apoptosis. It is most likely that RBCs are most labile as a result of HK-I/R deficiency since the HK-I gene but not the other isozyme genes are expressed in fetal and adult RBCs., (Copyright 2002 S. Karger AG, Basel)