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).     

Glucose‐6‐phosphate dehydrogenase deficiency: an unusual cause of acute jaundice after paracetamol overdose

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is the commonest human enzyme defect causing haemolytic anaemia after exposure to specific triggers. Paracetamol‐induced haemolysis in G6PD deficiency is a rare complication and mostly reported in children. We report the first case (to the best of our knowledge) of acute jaundice without overt clinical features of a haemolytic crisis, in an otherwise healthy adult female following paracetamol overdose, due to previously undiagnosed G6PD deficiency. It is important that clinicians consider this condition when a patient presents following a paracetamol overdose with significant and disproportionate jaundice, without transaminitis or coagulopathy.     

Glucose-6-phosphate dehydrogenase deficiency

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.     

Congenital haemolytic anaemia associated with adenylate kinase deficiency

SUMMARY. Chronic haemolytic anaemia associated with adenylate kinase (AK) deficiency is very rare and only seven cases in five families have been described. We present six children of one family who are deficient of this enzyme and in three of them a combined G6PD deficiency was found. AK deficiency was transmitted by an autosomal recessive gene and heterozygous state was not accompanied by disease, whereas homozygously affected individuals present a congenital chronic non-spherocytic haemolytic anaemia with haemoglobin levels of 8-9 g/dl. Patients also deficient in G6PD suffer from a more severe haemolytic anaemia with haemoglobin levels around 6 g/dl. The AK-deficient children are also mentally retarded. Splenectomy performed in five of the six patients resulted in complete remission of the haemolytic process.     

Two new mutations of the glucose-6-phospate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships

In two unrelated Spanish males with glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid^{406 C→T} and G6PD Madrid^{1155 C→G} .
In vitro biochemical characterization of both mutant enzymes showed important differences in their molecular properties according to their different clinical behaviour. In G6PD Valladolid, the mutation of which is located in exon 5, the normal in vitro heat stability may explain its mild clinical expression (low-grade haemolysis interrupted by an acute haemolytic crisis at age 70). In G6PD Madrid, the mutation, located in exon 10, results in a deficient variant associated with neonatal jaundice and life-long chronic nonspherocytic haemolytic anaemia (CNSHA). This finding further emphasizes the importance of this specific region of the G6PD gene in the stabilization of the G6PD molecule. Putative relationships between these single point mutations and the molecular properties of the mutant enzymes are also discussed.     

Glucose-6-phosphate dehydrogenase (G6PD) deficiency--a cause of anaemia in pregnant women

Glucose-6-phosphate dehydrogenase (G6PD) is one of the most important cytoprotective enzymes for oxidative stress. The WHO classification of G6PD deficiency, based on enzyme activity and clinical significance, distinguishes five variants. Chronic haemolytic process is rare and the main factors causing haemolysis are: infections, substances derived from plants, drugs with high oxidation-reduction potential, stress, ketoacidosis in diabetes and surgery operations. We report two cases of women belonging to the class 3 of the WHO classification in whom haemolysis occured during pregnancy. One of the patients developed two incidents of haemolytic anaemia. The cause of the first episode, nine months before pregnancy, was probably infection of the urinary tract caused by Escherichia coli, but the influence of the drugs also cannot be excluded. Because of the genetic background of this enzymopathy we also examined members of the patients, families but did not find any evidence of G6PD deficiency among them. The reported cases indicate that haemolytic anaemia caused by G6PD deficiency may occur during pregnancy what can lead to many not only haematological but also serious obstetrical complications such as infertility, fetus malformations and even its death. We also draw attention to several difficulties in diagnosing G6PD deficiency especially during haemolysis.     

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 beta + alpha domain involved in dimerization.     

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

Summary .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 Union^1360T (two cases), G6PD Mediterranean^563T, (one case) and G6PD Aures^143c (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 12861 recognition site which enabled us to rapidly detect it by PCRRE 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 (3 8 5) 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 He substitution, thus confirming that G6PD Clinic is a new class I variant.     

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 Km for substrates and a reduced thermostability are common.     

Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.     

Definition of the mutations of G6PD Wayne, G6PD Viangchan, G6PD Jammu, and G6PD 'LeJeune'.

We report the nucleotide (nt) substitutions of four unrelated glucose-6-phosphate dehydrogenase (G6PD)-deficient males. Only the mutation of G6PD Wayne was unique. It was a nt 769 C----G substitution causing a deduced substitution of glycine for arginine at amino acid 257. This mutation is in a region in which G6PD mutations have previously been associated with chronic hemolytic anemia. The mutation of G6PD Jammu and G6PD Viangchan were identical: a G----A mutation at nucleotide 871, predicting a Val----Met substitution at amino acid 291. However, these two variants differ with respect to the 1311 polymorphism, suggesting that they may have arisen independently. Enzyme from a child with chronic hemolytic anemia, designated G6PD 'LeJeune', proved to be due to a G----T substitution at nt 637, a change identical with that in 3 unrelated patients who had been reported previously as having G6PD Gastonia, Minnesota and Marion. These findings support the suggestion that both polymorphic and sporadic G6PD deficiency mutations in unrelated persons with G6PD deficiency are often the same, even when thought to be distinct on the basis of biochemical characterization.     

The identification of a recurrent phosphoglycerate kinase mutation associated with chronic haemolytic anaemia and neurological dysfunction in a family from USA

Phosphoglycerate kinase (PGK) deficiency is a rare X-linked disease that is characterised by mild to severe haemolytic anaemia, rhabdomyolysis, and variable defects in the central nervous system. In a white American family, two sons presented with haemolytic anaemia, seizures, and developmental delay. The diagnosis of PGK deficiency was made based on the remarkably low (<5% of normal) erythrocyte PGK enzyme activity level and the identification of a missense (c. 491A --> T) PGK1 gene mutation. This mutation results in an Asp164Val amino acid substitution, which has previously been designated PGK-Amiens and PGK-New York. The two new patients have the full clinical syndrome of PGK deficiency including haemolytic anaemia, developmental delay and seizures, and in the proband, hemiplegic migraines, retinal dystrophy and muscle fatigue. The PGK-Amiens/New York mutation had previously been found in a French patient and also in a large Chinese-Australian kindred, indicating that either the c. 91A --> T mutation is a recurrent mutation or that there is shared ancestry between the patients that have been identified so far with the mutation. Haplotype analysis of the c. 91A --> T mutation indicated that this was a recurrent mutation.     

Two new phosphoglycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain

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.     

Glucose-6-phosphate dehydrogenase Guadalajara--a case of chronic non-spherocytic haemolytic anaemia responding to splenectomy and the role of splenectomy in this disorder

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.     

G6PD deficiency: a classic example of pharmacogenetics with on‐going clinical implications

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.     

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.     

Molecular basis of chronic non-spherocytic haemolytic anaemia: a new G6PD variant (393 Arg→His) with abnormal KmG6P and marked in vivo instability

Summary. More than 80 genetic variants of glucose-6-phosphate dehydrogenase (G6PD) are associated with chronic non-spherocytic haemolytic anaemia (CNSHA). In order to help clarify the molecular basis of this association, we have carried out a detailed biochemical and genetic characterization of two G6PD deficient brothers affected by CNSHA. The G6PD from the two patients has altered electrophoretic mobility, abnormally elevated Michaelis constant ( K _m) for G6P, and extreme instability in vivo and in vitro. By comparison with published information we found that this is a new G6PD variant which we have designated G6PD Portici. The entire coding region of the gene has been sequenced, and a single point mutation, a G→A transition, was found at position 1178 in exon X, causing a substitution of histidine for arginine at residue 393 in the polypeptide chain. By polymerase chain reaction (PCR) amplification followed by diagnostic restriction enzyme analysis and allele-specific oligonucleotide hybridization we have demonstrated the inheritance of this mutation in the patient's family. Our results support the notion of a causative link between this mutation in the G6PD gene and CNSHA. Our data, in combination with previous data in the literature, suggest that the three-dimensional structure of G6PD is such as to cause interaction in the binding of its two substrates, G6P and NADP.     

Glucose 6-phosphate dehydrogenase deficiency and red cell membrane defects: additive or synergistic interaction in producing chronic haemolytic anaemia

Summary. We have investigated two unrelated patients with congenital haemolytic anaemia in both of whom we found a combination of hereditary spherocytosis (HS) and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Segregation of the two defects was documented in both families, who had different molecular abnormalities for both HS and G6PD deficiency. In one family the propositus had a reduced level of spectrin and G6PD Seattle (282Asp His). In the other family the propositus had a band 3 abnormality and was heterozygous for G6PD Mediterranean (188Ser Phe). From a comparison of clinical and haematological findings in family members with either or both abnormalities we conclude that in one case the two defects exhibited a synergistic effect, resulting in a severe chronic haemolytic anaemia; whereas in the other the association was simply additive.     

Molecular basis of G6PD deficiency in India

G6PD deficiency has been reported from India more than 30 years ago and about 13 variants have been characterized biochemically. Here, we report the results of an epidemiological study investigating G6PD deficiency and the mutations among 14 heterogenous populations of India. Of the 3166 males tested, 332 (10.5%) were found to be G6PD-deficient and the prevalence rate varied from 5.7% to 27.9% in the different population groups. Molecular characterization revealed that G6PD Mediterranean (563 C-->T) was the commonest (60.4%) deficient variant followed by G6PD Kerala-Kalyan (949 G-->A; 24.5%) and G6PD Orissa (131 C-->G; 13.3%). G6PD Mediterranean had a more widespread distribution as compared to G6PD Kerala-Kalyan and G6PD Orissa and was associated with both 1311 C and 1311 T polymorhism. G6PD Mediterranean was found to have significantly lower red cell enzyme activity and more severe clinical manifestations than the other two. G6PD Chatham (1003 G-->A) with undetected red cell enzyme activity and G6PD Insuli (989 G-->A) with normal G6PD activity were very rare in the Indian population. The absence of a large number of mutations causing G6PD deficiency points to the fact that the genetic diversity of these populations is considerably lowered than expected.     

Congenital dyserythropoietic anaemia type II associated with a new type of G6PD deficiency (G6PD Gabrovizza)

A 6-year-old boy with chronic haemolytic anaemia was found to have glucose 6-phosphate dehydrogenase (G6PD) deficiency and the morphological, ultrastructural and serological features of congenital dyserythropoietic anaemia (CDA) type II. The patient's mother was heterozygous for G6PD deficiency. G6PD from the patient's red cells, upon partial purification and full characterization, was found to be a new variant designated G6PD Gabrovizza. We conclude that two distinct genetic abnormalities coexisted in this patient. We suggest that CDA type II may become clinically more expressed when another abnormality of the erythrocytes coexists.