Chronic Nonspherocytic Hemolytic Anemia Research Articles

Prevalência da deficiência da glicose-6-fosfato desidrogenase em doadores de sangue de Mossoró, Rio Grande do Norte

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. It affects as many as 330 million individuals worldwide. This deficiency may determine neonatal jaundice, chronic nonspherocytic hemolytic anemia and acute hemolytic anemia induced by drugs, infections and broad bean ingestion. The efficacy of blood transfusion is decreased when the donor is G6PD deficient. In this study, we aimed at determining the prevalence of G6PD deficiency in blood donors of Mossoro, Brazil. Samples of 714 blood donors (576 men and 138 women; 343 white and 371 non-white) with ages ranging from 18 to 62 years and that accepted to participate in the study were analyzed. All participants answered a standard questionnaire. G6PD activity was analyzed by the methemoglobin reduction test with deficiency being confirmed by the semiquantitative test. The overall prevalence of G6PD deficiency in blood donors was 3.8%, similar to the rate described for others regions of Brazil. There was no significant statistical difference in the frequency of G6PD deficiency between men and women, nor between white and non-white blood donors. This relatively high frequency of G6PD deficiency highlights a need to screen blood donors for this condition.

Rescue of Pyruvate Kinase Deficiency in Mice by Gene Therapy Using the Human Isoenzyme

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is a disorder caused by mutations in the PKLR gene that produces chronic nonspherocytic hemolytic anemia. Besides periodic blood transfusion and splenectomy, severe cases require bone marrow (BM) transplant, which makes this disease a good candidate for gene therapy. Here, the normal human R-type pyruvate kinase (hRPK) complementary (cDNA) was expressed in hematopoietic stem cells (HSCs) derived from pklr deficient mice, using a retroviral vector system. These mice show a similar red blood cell phenotype to that observed in human PKD. Transduced HSCs were transplanted into myeloablated adult PKD mice or in utero injected into nonconditioned PKD fetuses. In the myeloablated recipients, the hematological manifestations of PKD were completely resolved and normal percentages of late erythroid progenitors, reticulocyte and erythrocyte counts, hemoglobin levels and erythrocyte biochemistry were restored. Corrected cells preserved their rescuing capacity after secondary and tertiary transplant. When corrected cells were in utero transplanted, partial correction of the erythrocyte disease was obtained, although a very low number of corrected cells became engrafted, suggesting a different efficiency of cell therapy applied in utero. Our data suggest that transduction of human RPK cDNA in PKLR mutated HSCs could be an effective strategy in severe cases of PKD.

A Somatic Mutation in the G6PD Gene in Hematopoietic Cells Causes a Chronic Haemolytic Anemia.

Abstract 4033Poster Board III-969G6PD deficiency is the more common human enzyme defect, leading typically to an acute intravascular hemolysis occurring when red cells are exposed to an oxidative stress. However, in rare patients, very low enzymatic level induces the class I G6PD deficiency according to the WHO classification, i.e. a chronic non-spherocytic hemolytic anemia. These cases are all sporadic, occur worldwide, and almost all arise from de novo independent mutations. These mutations are found at the genomic level in both hematopoietic and non hematopoietic cells and occur recurrently in “hot spots”: most of them are located in the exon 10 of the G6PD gene, implicated in the dimer formation and the stability of the active enzyme. No null or frameshift mutations have been reported yet, probably because such mutations would be lethal; indeed, a minimal residual G6PD activity is essential during embryogenesis.We report here the case of 65 years-old Caucasian man referred in 1993 for hemolytic anemia. No personal or familial history of anemia was noted. Nine and five years before, the hemoglobin (Hb) level and the mean corpuscular volume (MCV) were normal. At diagnosis, the anemia was moderate (Hb: 11.9 g/dL), macrocytic (MCV: 104 fL) and regenerative (reticulocyte count: 550G/L) with hemolytic features. Platelets and leucocytes counts were normal. No clinical or ultrasound spleen enlargement was noted. The screening tests ruled out all the classical causes of acquired hemolytic anemia. Red cells half life after Cr51 labelling was shortened with autologous (5 days) but not with allogenous red cells confirming the corpuscular mechanism of the hemolysis. Dosage of erythocyte G6PD activity revealed a very low level (from 0.6 to 1.5 UI/g of Hb, normal range 5.3-7.9). Pyruvate kinase, pyrimidine 5'-nucleotidase and hexokinase enzymatic activities were increased in agreement with the reticulocytosis. With a follow-up of 16 years, evolution was marked by a progressive worsening of the anemia, requiring 8 to 16 packed red cell transfusions per year in parallel with an iron chelation and folic acid therapy. Concomittantly, the macrocytosis increased up to 144 fL.At the molecular level, sequencing of the genomic DNA of the G6PD gene revealed presence of a single nucleotide mutation that altered the IVS 10 nucleotide 1 G>A from donor consensus sequence, leading to an impaired splicing between exon 10 and exon 11. The missplicing creates a premature termination codon giving theorically a truncated protein (464 versus 514 amino-acid).This severe genotype was discordant with the normal hemoglobin level five years before the diagnosis. Moreover, such a mutation at the germinal level would be expected to be lethal; therefore, we hypothesized that it arised at the somatic level in an hematopoietic stem cell; we measured the G6PD activity in hematopoietic and non hematopoietic cells and observed that it was decreased in red cells, polymorphonuclear cells and lymphocytes from blood, but was normal in skin fibroblasts; molecular analysis confirmed that the mutation was present in blood mononuclear cells but was absent in the fibroblasts.Bone marrow CD34+ cells were then sorted and plated at one cell per dish in the presence of a cocktail of cytokines including EPO and the different clones were harvested at day 10 for genomic analysis of the G6PD gene. Out of the 44 tested clones, we found that most of them carried the mutation (61%), but we were still able to detect a few unmutated clones (7%), reflecting the persistence of a minor polyclonal unmutated hematopoiesis. Surprisingly, we detect in the rest of the clones both the normal and mutated genotypes; at this time, we have no clear explanation to this finding.Acquired modifications of structure or expression of genes implicated in red cell physiology have been long recognized, occurring principally in myeloid malignancies such as myelodysplastic syndromes (MDS). This includes genes implicated in the red cell membrane skeleton or in haemoglobin synthesis or structure. However, our patient presented no MDS features on repeated bone marrow examination and on cytogenetic analysis. Regardless to all our data, we concluded that our patient present the first case ever reported of chronic non spherocytic anemia related to an acquired somatic mutation of the G6PD gene at the level of a hematopoietic pluripotent cell. Disclosures:No relevant conflicts of interest to declare.

A Severe α Thalassemia Case Compound Heterozygous for Hb Adana in α1 Gene and 20.5 kb Double Gene Deletion

We report a 6-year-old boy diagnosed as transfusion dependent chronic nonspherocytic hemolytic anemia since 40 days old. Hemoglobin H inclusions were detected with brilliant cresyl blue preparation. His parents were found to be normal on physical examination. His mother had hemoglobin level of 9.34 g/dL, accompanied by typical thalassemic changes of the red cells, and inclusion bodies were also detected with brilliant cresyl blue staining. His father had normal hemoglobin level and borderline red cell indices. Mutation analysis using strip assay capable of detecting 22 mutations within the alpha genes was performed for the proband and the parents which revealed that the case was compound heterozygous for Hb Adana in alpha1 gene and 20.5 kb double gene deletion. The father was found to be heterozygous for Hb Adana alpha1 gene whereas the mother was found to be compound heterozygous for 20.5 kb double gene deletion and 3.7 kb single gene deletion. It is well known that non deletional forms of HbH disease are more severe than the deletional forms. This case represents another example of the nondeletional mutation underlying Hb Adana, which is rarely seen in alpha1 gene, and illustrates the distinctive phenotypes of both the deletional and nondeletional forms of hemoglobin H disease within the same family.

A novel G6PD mutation leading to chronic hemolytic anemia

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.

Chronic non-spherocytic hemolytic anemia associated with severe neurological disease due to -glutamylcysteine synthetase deficiency in a patient of Moroccan origin

A previously undescribed mutation of hereditary gamma-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage.

G6PD deficiency: the genotype-phenotype association

Deficiency of glucose-6-phosphate dehydrogenase is a very common X-linked genetic disorder though most deficient people are asymptomatic. A number of different G6PD variants have reached polymorphic frequencies in different parts of the world due to the relative protection they confer against malaria infection. People, usually males, with deficient alleles are susceptible to neonatal jaundice, and acute hemolytic anemia, usually during infection, after treatment with certain drugs or after eating fava beans. Very rarely de novo mutations can arise causing the more severe condition of chronic nonspherocytic hemolytic anemia. Altogether 160 different mutations have been described. The majority of mutations cause red cell enzyme deficiency by decreasing enzyme stability. The polymorphic mutations affect amino acid residues throughout the enzyme and decrease the stability of the enzyme in the red cell, possibly by disturbing protein folding. The severe mutations mostly affect residues at the dimer interface or those that interact with a structural NADP molecule that stabilizes the enzyme.

In VitroandIn VivoExpression of Human Erythrocyte Pyruvate Kinase in Erythroid Cells: A Gene Therapy Approach

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Pyruvate kinase deficiency: The genotype-phenotype association

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anaemia. The disease is transmitted as an autosomal recessive trait, clinical symptoms usually occurring in compound heterozygotes for two mutant alleles and in homozygotes. The severity of haemolysis is highly variable, ranging from very mild or fully compensated forms to life-threatening neonatal anaemia necessitating exchange transfusions. Erythrocyte PK is synthesised under the control of the PK-LR gene located on chromosome 1. One hundred eighty different mutations in PK-LR gene, mostly missense, have been so far reported associated to PK deficiency. First attempts to delineate the genotype-phenotype association were mainly based on the analysis of the enzyme's three-dimensional structure and the observation of the few homozygous patients. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined. However, the clinical manifestations of red cell enzyme defects are not merely dependent on the molecular properties of the mutant protein but rather reflect the complex interactions of additional factors, including genetic background, concomitant functional polymorphisms of other enzymes, posttranslational or epigenetic modifications, ineffective erythropoiesis and differences in splenic function.

Red cell glucose phosphate isomerase (GPI): a molecular study of three novel mutations associated with hereditary nonspherocytic hemolytic anemia

Molecular characteristics of red blood cell (RBC) glucose phosphate isomerase (GPI) deficiency are described in two Spanish patients with chronic nonspherocytic hemolytic anemia. One patient, with residual GPI activity in RBCs of around 7% (GPI-Catalonia), is homozygous for the missense mutation c.1648A>G (p.Lys550Glu) in exon 18. The other patient, with residual activity in RBCs of around 20% (GPI-Barcelona), was found to be a compound heterozygote for two different missense mutations: c.341A>T (p.Asp113Val) in exon 4 and c.663T>G (p.Asn220Lys) in exon 7. Molecular modeling using the human crystal structure of GPI as a model was performed to determine how these mutations could affect enzyme structure and function.

A novel missense mutation in glucose-6-phosphate dehydrogenase gene causing chronic nonspherocytic hemolytic anemia in an Indian family

A novel missense mutation in glucose-6-phosphate dehydrogenase gene causing chronic nonspherocytic hemolytic anemia in an Indian family

Life-threatening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression

AbstractHuman erythrocyte R-type pyruvate kinase (RPK) deficiency is an autosomal recessive disorder produced by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Survival of patients with severe RPK deficiency has been associated with compensatory expression in red blood cells (RBCs) of M2PK, an isoenzyme showing wide tissue distribution. We describe a novel homozygous null mutation of the PKLR gene found in a girl with a prenatal diagnosis of PK deficiency. The mutant PK gene revealed an 11-nucleotide (nt) duplication at exon 8, causing frameshift of the PKLR transcript, predicting a truncated protein inferred to have no catalytic activity. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) detected no M2PK expression in the peripheral blood red cell fraction. The expression of mutant RPK mRNA in the RBCs was almost 6 times higher than that detected in a control patient with hereditary spherocytosis. This molecular phenotypic analysis of the null mutation in the PKLR gene provides evidence for a lack of M2PK in the mature RBCs of this patient and suggests that normal red cell functions and survival are achieved through a population of young erythroid cells released into the circulation in response to anemia. (Blood. 2005;106:1851-1856)

Red Cell Pyruvate Kinase Deficiency: Molecular Characterization of 10 New Variants.

PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table:PtOriginHb (g/dL)Tx (n.)PK Activity (IU/gHb)MutationAminoacidic substitutionSDItaly15.1010.6107G / ?Ala36Gly/?TTAustralia8.90nd409A / del5006bp( IVS3-nt 1431)Ala137Thr / del ex 4-11NRItaly9>505.5661A /1209AAsp221Asn /Met403IleNAItaly9>505.3661A /1209AAsp221Asn /Met403IleSAItaly1205.51456T/ 1209AArg486Trp/ Met403IleSCItalyndnd5.61529A/ 859CArg510Gln/ Phe287LeuCMItaly9.5013.61456T/ 958AArg486Trp/ Val320MetPSItaly13.2081094T /?Lys365Met /?VRItaly10.5010.71706A / ?Arg569Gln /?GRGuinea14.407.61269A/ IVS9+43cSplice site/?Ref. Values12.2–1611.1–15.5Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln.A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.

Glucose phosphate isomerase deficiency: enzymatic and familial characterization of Arg346His mutation

Homozygous glucose phosphate isomerase (GPI) deficiency is one of the most important genetic disorders responsible for chronic non-spherocytic hemolytic anemia (CNSHA), a red blood cell autosomal recessive genetic disorder which causes severe metabolic alterations. In this work, we studied a patient with CNSHA due to an 82% loss of GPI activity resulting from the homozygous missense replacement in cDNA position 1040G>A, which leads to substitution of the protein residue A346H mutation. The enzyme is present in a dimeric form necessary for normal activity; the A346H mutation causes a loss of GPI capability to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism.

Red Cell Disorders

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme of the pentose phosphate shunt pathway a major function of which is to prevent cellular oxidative damage. Deficiency in red blood cells is associated with a number of varied clinical manifestations. Chronic non-spherocytic haemolytic anaemia is uncommon but is usually characterized by chronic haemolysis, often with severe anaemia. In the past splenectomy in this condition has been thought to be of questionable benefit. We report a case of G6PD Guadalajara where splenectomy produced transfusion independence and have reviewed the literature. Those cases with exon 10 mutations often have a severe clinical phenotype, which responds to splenectomy. This procedure should be considered in this condition.

Glucose-6-phosphate dehydrogenase deficiency in India.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest red cell enzymopathy in humans and has an X-linked inheritance. It has been reported from India more than 30 years ago and the prevalence varies from 0-27% in different caste, ethnic and linguistic groups. The major clinical manifestations are drug induced hemolytic anemia, neonatal jaundice and chronic non-spherocytic hemolytic anemia. Individuals with G6PD deficiency have a selective advantage against falciparum malaria. Thirteen biochemically characterized variants have been reported from India. At the molecular level, G6PD Mediterranean is the most common deficient variant in the caste groups whereas, G6PD Orissa is more prevalent among the tribal of India. The third common variant seen in India is G6PD Kerala-Kalyan.

A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia—characterization of enzyme using yeast expression system and molecular modeling

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia. We studied a novel point mutation in the G6PD gene 1226 C→G, predicting the proline 409 to arginine substitution (G6PD Suwalki). We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant. We showed that human wild-type, as well as the mutated (1226 C→G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene). Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the K m values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P. The P409R substitution leads to drastic changes in G6PD kinetics. The specific activity as well as stability of mutated G6PD is also significantly reduced. Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme. The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP +.

Distinct phenotypic expression of two de novo missense mutations affecting the dimer interface of glucose-6-phosphate dehydrogenase

Mutations encoding class I glucose-6-phosphate dehydrogenase (G6PD) variants are associated with chronic nonspherocytic hemolytic anemia (CNSHA), the most severe phenotypic expression of G6PD deficiency. These mutations frequently affect the G6PD dimer interface that is essential for enzymatic activity. We detected two de novo missense mutations concerning residues located close together in the dimer interface in two patients with severe G6PD deficiency. A novel c.1225C>T missense mutation was identified in a male neonate who presented with hemolysis and severe hyperbilirubinemia and the predicted Pro409Ser substitution constituted a novel class I variant, designated G6PD Utrecht. G6PD deficiency in the second patient was due to the once previously reported class I variant G6PD Sumaré (Val431Gly). Structural analysis revealed that the mutated residues Pro409 and Val431, located on different subunits, interact directly across the subunit interface and perturb formation of the G6PD dimer upon mutation. Favism and mild chronic hemolysis characterized the phenotype of the patient with G6PD Sumaré which contrasts with the more severe clinical picture of the patient with G6PD Utrecht and, in addition, that of the patient originally described with G6PD Sumaré. We postulate that this G6PD variant is at the crossing between class I and class II G6PD deficiency and its ultimate phenotypic expression is either aggravated or ameliorated by other (extra)genetic factors.

Red cell adenylate kinase deficiency: molecular study of 3 new mutations (118G>A, 190G>A, and GAC deletion) associated with hereditary nonspherocytic hemolytic anemia

AbstractWe report here 2 patients with chronic nonspherocytic hemolytic anemia (CNSHA) and severe red blood cell (RBC) adenylate kinase (AK) deficiency. One of these patients, a boy of Spanish origin, exhibited a neonatal icterus and splenomegaly and required blood transfusions until the age of 2 years. The other patient was a white, American infant born to parents who were first cousins; he also presented with neonatal icterus and anemia. In neither case was psychomotor impairment observed. The first patient was found to be a compound heterozygote for 2 different missense mutations, 118G>A(Gly40Arg) and 190G>A(Gly64Arg) (cDNA sequence first described by Matsuura et al, 1989). The second patient was homozygous for an in-frame deletion (GAC) from nucleotide (nt) 498 to 500 or nt 501 to 503 of the cDNA sequence, predicting deletion of either aspartic acid (Asp) 140 or 141. The crystal structure of porcine cytosolic AK was used as a molecular model to investigate how these mutations may affect enzyme structure and function. (Blood. 2003;102:353-356)

Mild hemolysis in a girl with G6PD Sumaré (class I variant) associated with G6PD A−

In the present study we describe the clinical and laboratory features of a female child, a compound heterozygote for glucose-6-phosphate dehydrogenase (G6PD) Sumaré (1292T→G) and African variants (202G→A). G6PD Sumaré is a variant causing chronic nonspherocytic hemolytic anemia. The child had neonatal jaundice 2 days after birth and needed phototherapy for 8 days. Since then, she has not had episodes of dark urine or new episodes of jaundice. She has not had hemolytic crises in spite of five respiratory infections and antibiotics administration. Laboratory data showed a reticulocytosis (5.6%) without anemia and serum unconjugated bilirubin at the upper limit of the normalcy. No hemoglobin and hemosiderin in the urine were detected. G6PD activity at 37°C was 1.15 UI/g Hb and G6PD cellulose acetate electrophoresis at pH 9.0 revealed two bands, in equal amounts, with normal and faster migration, respectively. She was homozygous for the normal (TA)6(TA)6 repeat in the UGT1A1 promoter. We conclude that the association of G6PD Sumaré and G6PD A− gave rise to a very mild chronic hemolysis, and the red cell population containing G6PD A− is probably enough to protect against severe chronic hemolysis.