Chronic Nonspherocytic Hemolytic Anemia Research Articles

Glucose-6-phosphate dehydrogenase deficiency, neonatal hyperbilirubinemia and Gilbert syndrome

The aim of this work was to evaluate the influence of abnormal UDP-glucoronosyltransferase-1 (UGT1A1) gene variant, on the incidence and severity of neonatal hyperbilirubinemia, in glucose-6-phosphate dehydrogenase (G6PD) deficient newborns. The A(TA)nTAA region in the promoter of the UGT1A1 gene was analysed in 20 children with G6PD deficiency. Fourteen of these children had the African type variant (G6PDA-) and 6 had different variants (G6PDNara, G6PDGuadalajara, G6PDDurham, G6PDTomah, G6PDAveiro e G6PDNashville) related to chronic nonspherocytic haemolytic anaemia (CNSHA). The existence of a positive history of neonatal hyperbilirubinemia, as well as its severity was registered. The incidence of neonatal hyperbilirubinemia was increased in this group of children (90%) and was not associated with abnormal alleles of the UGT1A1 gene. It was not possible to assess the influence of abnormal alleles in the severity of the neonatal hyperbilirubinemia. However, these abnormal alleles did not account for the severity of jaundice in children who presented variants related to CNSHA, since 5 were treated with an exchange transfusion and none presented abnormal alleles.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

AbstractMany mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Chronic non-spherocytic haemolytic disorders associated with glucose-6-phosphate dehydrogenase variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, being present in over 400 million people world wide. In a small number of cases, G6PD deficiency can lead to mild-to-severe chronic haemolysis, which is further exacerbated by oxidative stress. Such G6PD variants have been described all over the world and are responsible for chronic non-spherocytic haemolytic anaemia (CNSHA). To date 61 G6PD molecular variants associated with CNSHA have been identified, only some of which can cause the severe reduction in stability of the red blood cell enzyme. The distribution of the different mutations shows a predominance of small mutational events, and many have been found repeatedly in different parts of the world. By revisiting the 61 class I variants described so far, we can observe that a low inhibition constant (Ki) for NADPH, a higher Kmfor substrates and a reduced thermostability are common.

Glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase (G6PD) is expressed in all tissues, where it catalyses the first step in the pentose phosphate pathway. G6PD deficiency is prevalent throughout tropical and subtropical regions of the world because of the protection it affords during malaria infection. Although most affected individuals are asymptomatic, there is a risk of neonatal jaundice and acute haemolytic anaemia, triggered by infection and the ingestion of certain drugs and broad beans (favism). A rare but more severe form of G6PD deficiency is found throughout the world and is associated with chronic non-spherocytic haemolytic anaemia. Many deficient variants of G6PD have been described. DNA sequence analysis has shown that the vast majority of these are caused by single amino acid substitutions. The three-dimensional structure of G6PD shows a classical dinucleotide binding domain and a novel β+α domain involved in dimerization.

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP+ molecule and provides insights into enzyme deficiency

Background: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer↔tetramer equilibrium and its stability is dependent on NADP+ concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia.Results: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459→Leu) at 3 Å resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP+ molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer–dimer contacts making the tetramer are charge–charge interactions.Conclusions: The importance of NADP+ for stability is explained by the structural NADP+ site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP+ and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5′ nucleotidase deficiency: 25 years later

In 1972, Valentine et al described, under the name of ‘non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency’, an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden–Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of ‘haemolytic disorders associated with increased ATP’. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5′ nucleotidase (P5N). In conclusion, all these cases were formerly referred to as ‘high ATP syndromes’ because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

Biochemical and genetic basis of red cell enzyme deficiencies

Enzyme deficiencies have been identified in all erythrocyte pathways. Their frequencies differ with respect to the affected enzyme, the severity of the clinical manifestations and the geographical distribution. Most mutations are found within the coding sequences of genes, missense mutations occurring more often than deletions, insertions, splice site defects or premature stop codons. Promoter mutations are rare. The clinical manifestations are chronic or non-chronic haemolytic anaemias. The first of these are characterized by an impairment of cell function at normal values of the external load parameterskATPase and kGSHox. Haemolysis with a non-chronic course is induced only at enhanced values of the load parameters, caused by free radical generation by oxidative drugs, fava beans, infections, fever and physical exercise. The development of secondary haemochromatosis is the most common cause of mortality in patients suffering from severe chronic non-spherocytic haemolytic anaemia. Intracellular iron deposits must be prevented by timely treatment with effective chelating agents.

Several mutations including two novel mutations of the glucose-6-phosphate dehydrogenase gene in Polish G6PD deficient subjects with chronic nonspherocytic hemolytic anemia, acute hemolytic anemia, and favism

DNA sequencing revealed seven different glucose-6-phosphate dehydrogenase (G6PD) mutations in G6PD deficient subjects from 10 Polish families. Among them we found two novel mutations: 679C-->T (G6PD Radlowo, class 2) and a 1006A-->G (G6PD Torun, class 1). Variant G6PD Radlowo was characterized biochemically. Both novel mutations were analyzed using a model of the tertiary structure of the human enzyme. The main chain of G6PD Torun is different from the wild-type G6PD. The remaining mutations identified by us in deficient Polish patients were: 542A-->T (G6PD Malaga), 1160G-->A (G6PD Beverly Hills), 1178G-->A (G6PD Nashville), 1192G-->A (G6PD Puerto Limon), and 1246G-->A (G6PD Tokyo). Variant Tokyo was found in four families. In one of them favism was the first clinical sign of G6PD deficiency and chronic nonspherocytic hemolytic anemia (CNSHA) was diagnosed later. Variants G6PD Nashville and G6PD Puerto Limon were accompanied by the silent mutation 1311C-->T of the G6PD gene.

Amino acid conservation and clinical severity of human glucose-6-phosphate dehydrogenase mutations.

More than a hundred naturally occurring mutations of human glucose-6-phosphate dehydrogenase (G6PD) have been identified at the amino acid level. The abundance of distinct mutation sites and their clinical manifestations make this enzyme ideal for structure-function analysis studies. We present here a sequence and structure combined analysis by which the severity of clinical symptoms resulting from point mutations of this enzyme is correlated with quantified degrees of amino acid conservation within 23 G6PD sequences from different organisms. Our analysis verifies, on a quantitative basis, a widely held notion that clinically severer mutations of G6PD usually occur at conserved amino acids. However, marked exceptions to this general trend exist which are most notably revealed by a number of mutations associated with chronic nonspherocytic hemolytic anemia (class I variants). When mapped onto a homology-derived structural model of human G6PD, these class I mutational sites of low amino acid conservation appear to localize in two spatially distinct clusters, both of which are populated with mutations consisting mainly of clinically severer variants (i.e. class I and class II). These results of computer-assisted analyses contribute to a further understanding of the structure-function relationships of human G6PD deficiency.

Clinical and haematological consequences of recurrent G6PD mutations and a single new mutation causing chronic nonspherocytic haemolytic anaemia.

We have determined the causative mutation in 12 cases of glucose-6-phosphate dehydrogenase deficiency associated with chronic non-spherocytic haemolytic anaemia. In 11 of them the mutation we found had been previously reported in unrelated individuals. These mutations comprise seven different missense mutations and a 24 base pair deletion. G6PD Nara, previously found in a Japanese boy. Repeated findings of the same mutations suggests that a limited number of amino acid changes can produce the CNSHA phenotype and be compatible with normal development. The one new mutation we have found, G6PD Serres, is 1082 C-->T causing a 361 Ala-->Val substitution in the dimer interface where most other severe G6PD mutations are found. Now that several patients with the same mutation have been reported we can compare the resulting clinical phenotypes. For each mutation we find a reasonably consistent clinical picture, ranging from mild (G6PD Clinic) through moderate (G6PD Nashville) to severe (G6PD Beverly Hills and G6PD Nara).

Index of Suspicion

Index of Suspicion

Glucose-6-phosphate dehydrogenase Durham: A de novo mutation associated with chronic hemolytic anemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked enzyme defect. We report a new variant, G6PD Durham 713G , that is associated with chronic nonspherocytic hemolytic anemia. The G6PD Durham 713G variant has a unique biochemical and enzymatic profile and a novel A→G substitution mutation at nucleotide 713, changing lysine to arginine at amino acid 238. This mutation was not found in the mother of our patient, indicating that G6PD Durham 713G resulted from a de novo mutation. (J Pediatr 1997;131:284-7)

Congenital 6-phosphogluconate dehydrogenase (6PGD) deficiency associated with chronic hemolytic anemia in a Spanish family

Clinical and metabolic studies were performed in four members of a Spanish family with partial (50%) 6 phosphogluconate dehydrogenase (6PGD) deficiency. In all cases the activities of 6 phosphogluconolactone (6PGL) and glutathione reductase (GR) were normal, and the molecular characterization performed in the partially purified 6PGD from the propositus showed normal kinetic and electrophoretic patterns. Two females (the propositus and her sister) suffered from a well-compensated chronic nonspherocytic hemolytic anemia (CNSHA) and exhibited decreased RBC glutathione (GSH) stability with increased oxidative susceptibility, defined by enhanced malonyldialdehyde (MDA) generation "in vitro." The other two members of the family (the propositus's mother and brother) were clinically asymptomatic. In the propositus and her sister, RBC metabolism exhibited a markedly abnormal concentration of glycolytic intermediates, mainly characterized by striking increases in fructose 1,6 bisphosphate (50-fold), dihydroxiacetone-phosphate (20-fold) and glyceraldehyde 3-phosphate (tenfold). Although the precise mechanism of the hemolysis in the two patients is unknown, the enhanced oxidative threat observed in their RBCs may interfere in some way with the glycolytic pathway function, leading to a marked increase in certain metabolic intermediates located before the glyceraldehyde 3 phosphate dehydrogenase (GA3PD) step. Since it seems that GA3PD half-life is modulated by fluctuations of the cytosolic redox status, an "in situ" approach was simulated by using permeabilized RBCs. In these conditions, GA3PD activity was significantly lower in the propositus and her sister than in the asymptomatic members of the family and the simultaneous normal control.

Hematologically Important Mutations: Red Cell Pyruvate Kinase

Luciano Baronciani, Paola Bianchi, Alberto ZanellaPyruvate kinase (PK) deficiency is known tobe the most common cause of chronic nonsphero-cytic hemolytic anemia (CNSHA). Two PK genesare present in mammals, PKLR and PKM, but onlythe former codes for the isoenzyme normallyexpressed in the red cell (R-type).A total of 82 mutations in the PKLR geneassociated with CNSHA are here reported inTable 1, whereas Table 2 provides the poly-morphic sites identified in the gene. Mutationsand polymorphisms are designated using thecDNA sequence of the R-type isoenzyme andgenomic DNA sequence of the PKLR gene, withthe A of the initiation ATG being assigned num-ber 11. The GenBank accession numbers areD10326 D90465 for the cDNA and U47654for the genomic DNA. The numbering system inthe tables varies from that of GenBank and inorder to designate the initiated A as 11itisnecessary to subtract 39 from the GenBank se-quences.Additional information can be found in thepublished reviews (1,2) and a continuously up-dated review can be found on the World Wide Webin OMIM at:http://www3.ncbi.nlm.nih.gov:80/htbinpost/Omim/dispmim?266200

Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5- oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Three Cases of Hereditary Nonspherocytic Hemolytic Anemia Associated With Red Blood Cell Glutathione Deficiency

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Structural and functional properties of mutant Arg203Pro from yeast phosphoglycerate kinase, as a model of phosphoglycerate kinase-Uppsala.

A pathological variant of human phosphoglycerate kinase, phosphoglycerate kinase-Uppsala, associated with chronic nonspherocytic hemolytic anemia has been found to differ from the normal enzyme by substitution of an arginine at position 206 (corresponding to position 203 in yeast) by a proline. In order to understand the structural and functional consequences of this mutation, the corresponding mutant in yeast phosphoglycerate kinase was constructed. The three-dimensional structure of this mutant was resolved at 2.9 A. Although the overall structure is not modified, small local changes were observed. The kinetic parameters of the mutant were not found to be greatly affected, the catalytic constant being lowered by only 10-20%. The most significant difference when compared with the wild-type enzyme is a decrease in stability by about 3 kcal/mol. The physiological implications of this instability are discussed.

Molecular genetics of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene.

In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain, we have analysed the G6PD gene in 11 unrelated Spanish G6PD-deficient males and their relatives by using the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with a direct PCR-sequencing procedure and PCR-restriction enzyme (RE) analysis. We have identified eight different missense mutations, six of which have been reported in previously described G6PD variants. In nine patients who had presented with acute favism we found the following mutations: G6PD A-376G-202A (four cases), G6PD Union1360T (two cases), G6PD Mediterranean563T (one case) and G6PD Aures143C (one case). In the remaining patient a novel A to G transition was found at nucleotide position 209 which has not been reported in any other ethnic group. This mutation results in a (70) Tyr to Cys substitution and the resulting G6PD variant was biochemically characterized and designated as G6PD Murcia. This new mutation creates a Bsp 1286I recognition site which enabled us to rapidly detect it by PCR-RE analysis. In two patients with chronic non-spherocytic haemolytic anaemia (CNSHA) we found the underlying genetic defects, as had been noted previously, to be located within a cluster of mutations in exon 10. One of them had the T to C transition at nucleotide 1153, causing a (385) Cys to Arg substitution, previously described in G6PD Tomah. The other, previously reported as having a variant called G6PD Clinic, has a G to A transition at nucleotide 1215 that produces a (405) Met to Ile substitution, thus confirming that G6PD Clinic is a new class I variant.

Triosephosphate isomerase deficiency: biochemical and molecular genetic analysis for prenatal diagnosis

Inherited deficiency of the glycolytic enzyme triosephosphate isomerase leads to a multisystem disorder characterized by progressive neuromuscular dysfunction, chronic nonspherocytic hemolytic anemia and increased susceptibility to severe infections. Most patients die within the first 6 years. We examined a family with severe triosephosphate isomerase deficiency. The 1-year-old index patient suffered from hemolytic anemia, neuromuscular impairment and pneumonias, with the necessity of intermitten mechanical ventilation. Triosephosphate isomerase activity in erythrocytes was reduced to about 20% of normal. Heat stability of the enzyme was strongly reduced; concentration of the physiological substrate, dihydroxyacetone phosphate was increased 20-fold due to the metabolic block. Direct sequencing of the triosephosphate isomerase gene revealed homozygosity for the formerly described GAG-->GAC-mutation changing 104 Glu-->Asp. During a 2nd pregnancy we examined a cord blood sample obtained in the 19th gestational week. The biochemical data on enzyme activity, heat stability of the enzyme and concentration of dihydroxyacetone phosphate were in the normal range. The molecular genetic analysis confirmed the presence of the normal triosephosphate isomerase alleles. Pregnancy was continued, resulting in the delivery of an unaffected, healthy newborn.