Glucose-6-phosphate dehydrogenase ... - Wiley Online Library

10 downloads 7468 Views 361KB Size Report
Keywords G6PD assay; favism; molecular test; diagnosis; pyruvate kinase ... ribose availability which is essential for production of nucleo- tide coenzymes, the ... a functional domain, because the removal of NADP molecule would affect the ...
IUBMB

Life, 61(1): 27–34, January 2009

Critical Review Glucose-6-phosphate Dehydrogenase Laboratory Assay: How, When, and Why? Angelo Minucci, Bruno Giardina, Cecilia Zuppi and Ettore Capoluongo Laboratory of Clinical Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome, Italy

Summary Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD-deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow-chart for the complete diagnostic approach of G6PD-deficiency. Ó 2008 IUBMB IUBMB Life, 61(1): 27–34, 2009 Keywords

G6PD assay; favism; molecular test; diagnosis; pyruvate kinase; G6PD/6PGD.

INTRODUCTION Glucose 6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme, which is critical in the redox metabolism of all aerobic cells. It catalyzes the first and rate-limiting step of the pentose phosphate pathway, finalized to the NADPH synthesis and to ribose availability which is essential for production of nucleotide coenzymes, the replication of nucleic acids and, therefore cell division (1) (Fig. 1). G6PD-deficient red blood cells (RBCs) have been deeply characterized, because the pentose phosphate pathway is the Received 15 July 2008; accepted 7 August 2008 Address correspondence to: Ettore Capoluongo, Laboratory of Clinical Molecular Biology, Department of Biochemistry and Clinical Biochemistry, Catholic University Largo F. Vito 1, Rome 00168, Italy. Tel: 0039 06-30154250. Fax: 0039 06-30156706. E-mail: [email protected] ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.137

unique source of NADPH, which enables RBCs to counterbalance the oxidative stress triggered by several oxidant agents preserving the reduced form of glutathione (GSH). Through glutaredoxin, GSH protects the sulfhydryl (SH) groups in hemoglobin and in the red cell membrane from oxidation. In normal RBCs the ratio between reduced and oxidized GSH is 100:1 (2). If NADPH concentrations cannot be maintained, as in G6PD deficiency, the GSH levels fall and oxidative damage occurs resulting in an acute hemolysis.

STRUCTURE OF G6PD PROTEIN G6PD is a dimer and each subunit contains a single active site. The active human enzyme exists in a dimer $ tetramer equilibrium pH-dependent (3). The bacterial enzyme does not contain a site for a structural NADP molecule, a position considered as crucial for human enzyme, both for activity alone and for the long-term G6PD stability in physiological conditions. Really, this site could be considered both a structural and a functional domain, because the removal of NADP molecule would affect the association and the conformation of each subunit. In fact, each NADP1 is entirely bound within one subunit and one of the NADP1 ligands, Asp421, is at the center of the dimer interface (4). Two conserved sequence motifs were apparent in the alignment: the completely conserved eight-residue peptide RIDHYLGK (residues 198–205) corresponding to the substrate-binding site and the dinucleotide-binding fingerprint GxxGDLx (residues 38–44). Finally, the coenzyme-binding domain involves the residues 31–200, with the fingerprint sequence GASGDLA (3). FAVISM: AN OLD BUT ALWAYS ACTUAL DISEASE G6PD deficiency is the commonest clinically significant enzymopathy in humans. More than 400 million people worldwide are affected by this condition which may determine: (a) favism; (b) drug-induced acute hemolytic anemia; (c) severe chronic nonspherocytic haemolytic anemia, (d) neonatal jaundice,

28

MINUCCI ET AL.

Figure 1. G6PD enzyme metabolic pathway: the glycolysis and the pentose phosphate cycle (hexose monophosphate shunt).

Figure 2. Distribution of the known point mutations within the coding region of the G6PD gene. Mutations associated with chronic hemolytic anemia (CHA) are clustered to exon 10, the coding region for amino acids involved in the G6PD dimer interface and to exon 13, coding region for amino acids involved in the interactions with the ‘‘Structural NADP’’.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE LABORATORY ASSAY

29

Table 1 Classification of G6PD deficiencies following the WHO recommendations Class

Residual enzymatic activity

Frequency and geographical distribution

Protein location

Clinical symptoms

II

\1% or not detectable \10%

Dimer interface Structural NADP Dimer interface NADP binding site

Rare and without a precise geographical distribution Frequent and distributed throughout the world

G6PD-Buenos Aires, G6PD Durham G6PD-Mediterranean G6PD-Cassano G6PD-Santamaria

III

10–60%

Scattered throughout the whole enzyme

Chronic hemolytic anemia Acute hemolytic anemia fava beans and drugdependent Occasionally acute hemolytic anemia

Very frequent in malaria areas

IV

60–90% normal activity [110% increased activity

Neutral protein site

Asymptomatic

Undefined frequency

Neutral protein site or promoter mutations

Asymptomatic

Undefined frequency

G6PD-A2 G6PD-Seattle G6PD-Canton G6PD-Rignano G6PD-Montalbano G6PD-Orissa Not reported

I

V

and (e) hemolytic anemia associated with viral or microbiological infections. The highest prevalence of G6PD deficiency mainly regards tropical Africa, the Middle East, tropical and subtropical Asia, Papua New Guinea, and various Mediterranean regions, for example Sardinia island. In these geographic areas, G6PD deficiency may represent a selective advantage due to the increased resistance to severe Plasmodium falciparum infection of the affected individuals (5). All patients with favism are G6PD deficient, but many G6PD-deficient individuals can regularly eat fava beans showing very heterogeneous clinical signs, sometimes at subclinical and/or undetectable levels. These finding suggest that several factors affect the clinical phenotype, including the health status, age, life style and, finally, the amount of fava beans ingested overtime. Hemolytic crises may also occur after eating fresh beans or, more rarely, after ingestion of dried or frozen beans, (6). It has been suggested that the glycosides divicine and isouramil are the components of the bean causing the hemolysis, even if the complete mechanisms, by which this effects are determined, are still not yet well defined. For this reason, the enzyme deficiency is a necessary, but not sufficient, condition leading to the hemolysis (7).

GENETICS G6PD-enzyme is encoded by a human X-linked gene (Xq2.8) consisting of 13 exons and 12 introns, spanning nearly 20 kb in total; the first exon is noncoding, while the remaining 12 range from 120 to 236 bp. G6PD gene is probably the most polymorphic locus in humans, with over 400 allelic variants known. These variants have been biochemically characterized

Type of mutations

based on: (a) the different residual enzyme activities, (b) electrophoretic mobility patterns, and (c) physicochemical (such as thermostability and chromatographic behavior) or kinetic (km for glucose-6-phosphate or NADPH, pH dependence, and utilization of substrate analogues) properties (8). G6PD variants are grouped into five classes (Table 1) based on WHO guidelines (9). All G6PD mutations determining an enzyme deficiency (Fig. 2) affect the whole coding sequence (10). About 160 mutations have been reported, most of which are single-base substitutions leading to amino acid replacements, while rarely a second mutations is present in cis. The G6PD A2 is a peculiar genotype determined by the concomitant presence of A376G plus G202A mutations. Although the nucleotide 202 substitution accounts for at least 95% of the G6PD A2 molecular variants in Africa, two less frequent second mutation sites have been identified at 680 and 968 nucleotides (11). A second uncommon genotype, namely G6PD-Santamaria, a Class II variant, is also determined by two concomitant mutations at the 376 and 524 nucleotides. This mutation was firstly found in Costa Rica and is relatively less frequent in Southern Italy (12). Small and in-frame deletions are exceptions; the absence of genetic mutations determining the complete abolishment of the G6PD functionality (such as large deletions, nonsense or frameshift mutations) suggests that complete absence of the G6PD enzyme is incompatible with life (13). In addition, no mutations in the residues inside the active or in the promoter sites are reported, while de novo mutations have been identified (14). Finally, also the noncoding G6PD sequence should be involved in determining G6PD-deficiency, such as for subjects carrying the silent polymorphism 1311 (C?T) in association with IVS XI 93 (T?C), known as NlaIII polymorphism.

30

MINUCCI ET AL.

The mechanism determining this deficiency is, unfortunately, still unknown (15). Notaro et al. (16) found a strict correlation between amino acid substitution causing G6PD deficiency and the score of evolutionary history of the different domains affected by these mutations. It has been proposed that the ‘‘natural selection’’ favors an enzyme with decreased, but with significant activity into the cells. To understand the molecular basis of G6PD deficiency, and to correlate genotype with phenotype, the various point mutations must be analyzed in their three-dimensional structures with computational analysis, followed by site-directed mutagenesis experiments, for a complete evaluation of mutants’ residual activity, stability, and dependence on NADP1 concentration.

FIRST QUESTION: HOW? Enzymatic Evaluation of G6PD Deficiency Following the WHO recommendations (8, 9), the diagnosis of G6PD deficiency utilizes universal tests, mainly based on the generation of NADPH from NADP. 1. Semiquantitative assays are the following: (a) fluorescent spot test, which is rapid, simple, sensitive, and inexpensive. This test can be used in Countries where G6PD deficiency is both frequent and malaria endemic, before starting treatment with antimalarial drugs, such as primaquine. A variant of the spot test, not requiring the use of an ultraviolet lamp, but a naked eye evaluation (17), allows the large population screening in the tropical areas; (b) and other screening tests are available: some of these, determine the NADPH concentration indirectly, by measuring the reduced methemoglobin levels produced after NADPH oxidation (18). This technique may be applicable also to intact cells, with a direct detection of the G6PD activity in individual RBCs, with 75% accuracy. (c) Finally, the Heinz body examination and GSH stability test may be employed to distinguish G6PD-deficient from normal individuals. 2. For the biochemical definitive diagnosis, a quantitative analysis of G6PD activity is mandatory. The G6PD activity quantitative spectrophotometric assay, is based on the evaluation of absorbance at 340 nm given by NADPH formation; the quantitative evaluation is made by adding a precise amount of hemolysate to an assay mixture containing the substrate (glucose-6-phosphate) and its cofactor NADP; the rate of NADPH generation is spectrophotometrically measured at wavelength of 340 nm (19). The G6PD activity is finally expressed as G6PD IU/RBCs and G6PD IU/hemoglobin ratios. In normal RBCs, the G6PD activity ranges from 7 to 10 IU/g Hb, when measured at 30 8C. Diagnostic issues can arise when G6PD activity is measured after or during an episode of acute hemolysis, or in the presence of a high blood reticulocyte count, being the reticulocytes’ activity about five times higher than that of old RBCs, result-

ing in a false negative result. Because protein synthesis is absent in RBCs, the activity of G6PD, and of other enzymes (for example, the Pyruvate Kinase), gradually decreases during RBCs aging, which will be selectively destroyed. For example, the activity of individuals carrying the G6PD A2 variant may be misclassified as normal, if the test is performed soon after a posthemolytic event. Other discrepancies may occur in the assessment of neonatal screening, because neonates have a young RBC population. Also the X-inactivation phenomenon deserves important consideration. First, as males are hemizygotes, they must be either normal or G6PD-deficient. By contrast, females, can be either normal or deficient (homozygous), or intermediate (heterozygous). Heterozygous females (HF) with extremely skewed X inactivation have activity ranging from hemizygote to normal. In fact, the presence of genetic mosaics represents the most important clinical implication for females. In fact, HF may show a hemizygote-like phenotype differently from other autosomal recessive enzyme deficiencies, where heterozygotes are asymptomatic, because cells, with an enzyme level close to 50% of normal, are unaffected. Therefore, G6PD deficiency may be expressed both biochemically and clinically in heterozygote, with different degrees of severity. During an acute hemolytic episode occurred in carrier females, both the fluorescent and quantitative tests should be within normal ranges, because they only measure the activity of the remaining normal surviving RBC population. If an acute intravascular hemolytic G6PD-dependent anemia is suspected, any potentially dangerous drugs must be discontinued and the test should be rerun 10–15 days after, or later, if the patient has been transfused. In these cases, genetic analysis or family study, when available, can improve the diagnostic tool. G6PD activity assay must be performed on deleucocyted blood, to eliminate interferences given by G6PD activity of white blood cells, especially in the presence of leukocytosis. The optimal anticoagulant for G6PD stability is K2-EDTA that ensures good conservation of blood cells; however, also ACD (citric acid 1 sodium citrate 1 dextrose), CPD (citrate phosphate 1 dextrose), sodium citrate, and lithium heparin could be used (20). In the presence of these anticoagulants, G6PD activity is stable at 4 8C up to 72 h (21). To prevent a reduction of the G6PD activity, all samples should be transported in heat-insulated envelopes and refrigerated transport containers (21). When blood is collected in tubes containing ammonium oxalate or fluoride oxalate anticoagulants, G6PD activity must be assayed within 12 h, keeping the sample at 4 8C until used (21). For a complete diagnostic assessment of the G6PD deficiency rate, the following laboratory parameters are also important: RBCs and reticulocyte counts, total and indirect plasma bilirubin, plasma iron and lactate dehydrogenase levels, serum aptoglobin and ferritin amounts, and finally, urine hemoglobin

GLUCOSE-6-PHOSPHATE DEHYDROGENASE LABORATORY ASSAY

31

concentration. In the presence of higher indirect bilirubin levels, the study of UGTA-1 gene promoter should be considered to exclude the concomitant presence of Gilbert Syndrome. Also the Pyruvate Kinase (PK) activity assay may provide additional guidance. The correct choice of reference intervals, selected considering gender, age, and the characteristic of each singular population, may be another important tool for a better laboratory practice.

Molecular Diagnosis The molecular analysis may be useful for population screening, family studies, or prenatal diagnosis. Molecular tests are particularly important for the analysis of G6PD HF. Several molecular methods have been developed: amplification refractory mutation system (22), gradient gel electrophoresis (DGGE) (23), probe melting curve (24), microarray (25), denaturing high-performance liquid chromatography (26), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (27), reverse dot blot assay (28), the single base extension assay (29), and finally restriction fragment length analysis performed by microcapillary chip electrophoresis (30). The identification of the specific G6PD mutations can better describe the clinical phenotype and provide additional epidemiological information regarding the different geographical distribution of the genetic variants. For a correct laboratory practice, the molecular diagnosis of G6PD deficiency should employ two analytical steps: 1) a first screening level, to research the most frequent mutations belonging to a specific geographical area. In this case, a PCR coupled to RFLP represents a rapid, valid, and reliable molecular screening approach (30); 2) a second level, based on the whole gene sequencing, finalized to the identification of the less frequent, or novel, mutations (Fig. 3). DNA-based test for the screening of the most frequent mutations in a specific geographical area can be used as an alternative tool to the biochemical assay. The costs for chemicals dedicated to molecular test are comparable to those used for the enzyme assay. In the future, more advances systems should be utilized to improve the efficiency of the molecular assay. Heterozygous Females G6PD HF should be early warned and, when necessary, treated as if they are G6PD deficient. None of the biochemical screening tests can reliably identify HF: thus, early individuation of female carriers of a defective G6PD variant, represent a very important diagnostic challenge. In fact, in steady state conditions, the screening test is low sensitive, because less than 46% of the deficient females results as abnormal at fluorescent semiquantitative biochemical test. Reclos and coworkers (31) reported that up to 60% of heterozygotes were not detected using the spectrophotometric G6PD assay alone. The use of a more sensitive enzymatic approach can improve the individuation of HF: in fact, measuring the G6PD and 6-

Figure 3. A molecular analysis strategy for the identification of G6PD mutations in Italian Population. The first step consists of the search most frequent mutations in Italy by PCR-RFLP. If at this level the result is negative, we sequence exons 3, 9, 10, 11, 12, and 13 for the identification of the less frequent mutations. In the presence of negative result we sequence the whole coding region and if necessary we analyze promoter, splicing junctions, and intronic mutations. Finally, we draw up a detailed report. phosphogluconate dehydrogenase (6PGD) activity separately, and then utilizing the ratio of the two enzymatic activities, an improvement of sensitivity of the biochemical tests should be obtained. In fact, the ratio determination allows to more accurately identifying the G6PD Mediterranean heterozygotes, reaching sensitivity and specificity values of about 85.3% and 97.4%, respectively (32). The relationship between these two enzymes is therefore an absolute measure of G6PD deficiency, which is not affected by the hemoglobin individual variations, RBC, reticulocytes, and leukocytes amounts. Our laboratory experience, based on the evaluation of more than 100 females screened by measuring the G6PD and 6PGD, allows to state that the results obtained with these two tests were not always concordant. In fact, the G6PD/6PDG ratio resulted more sensitive when compared with the classical test, allowing to address to the genetic analysis a sample of 17 females resulted negative at the classical quantitative analysis: these 17 females were

32

MINUCCI ET AL.

confirmed to be G6PD-mutation carriers. For these reasons, to improve the detection rate of possible mutation carrier females, we suggest to perform the G6PD/6PDG ratio determination. We underline that it is very important and the correct choice of the cut-off: we think that a value of \0.85 should permit the correct identification of all HF. In addition, this test should be of support for the recognition of Class IV deficient males, which may often result as normal when the classical quantitative test is performed. Also G6PD/PK ratio is more reliable than the G6PD activity alone, for the identification of G6PD heterozygotes, especially in patients with microcytic anemia. In fact the G6PD/PK ratio measurement, whose activities increase in the presence of microcytosis or a high number of young erythrocytes, respectively, is considered a useful diagnostic tool to identify the heterozygous G6PD-deficient individuals affected by the thalassemic trait or serum total iron deficiency (33). Another method for the detection of HF G6PD-deficient should be the cytochemical G6PD staining assay (34). Finally, because the preanalytical and analytical phases of the biochemical G6PD assay are crucial, these tests should be performed only by very specialized professionals, laboratories, or medical institutions. Before the test and after a casual discovering of a biochemical G6PD deficiency, a short interview with the G6PD-deficient individual or with its parents will be necessary for a complete clinical, laboratoristic, and genetic anamnesis. Finally, the training and updating of health professionals should be constantly encouraged.

SECOND QUESTION: WHEN DOES THE TEST NEED? In practical terms, testing for G6PD deficiency should be suggested when an acute hemolytic reaction, triggered by exposure to a known oxidative drugs, infections, or ingestion of fava beans occurs, either in children or in adults, particularly if they belong to African, Mediterranean, or Asian areas. Moreover, members (especially males) of families where jaundice, splenomegaly, or cholelithiasis are recurrent should be tested for G6PD deficiency. Newborn babies with severe prolonged neonatal jaundice, those belonging to Mediterranean or African ancestry in particular, are quite likely to have G6PD deficiency. Is the Neonatal Screening Advisable? The prenatal diagnosis of G6PD deficiency has been reported, although this approach is questionable when we consider the low mortality and morbidity related to G6PD deficiency. It is not clear why G6PD deficiency leads to an increased incidence of neonatal jaundice in both males and females (35): in the United States it has been estimated that a 30% of jaundiced infants who have permanent neurological damage (kernicterus) are G6PD deficient. In fact, G6PD mutations may contribute to the higher risk of neonatal hyperbilirubinemia and account for about 22% of all cases in the US Pilot Kernicterus Registry (36). Neonatal screening for G6PD defi-

ciency is routinely performed in many countries, mainly on dried blood spots, commonly using the semiquantitative method described by Beutler (37), or modifications to this technique (38). This assay discriminates between deficient (partially or totally) and normal case, but presents some disadvantages. The most important drawback of this method is its low cut off limit (\ 2.1 UI/g Hb) that it is too low for an accurate identification of all partially deficient HF (31). In fact it can only recognize the totally deficient individuals (\20% residual enzymatic activity), while it could erroneously classify, as normal, the partially deficient neonates (males and females with residual enzymatic activity between 20 and 60%) (39): because the missed HF might show G6PD-deficient phenotype later, the current G6PD neonatal screening might not be helpful in diagnosis or prevention of this disease in females. It is evident that further works should be done on larger population samples to determine highest cut offs for the correct neonatal classification at birth. Alternatively, a fully quantitative neonatal G6PD test should be mandatory, sometimes also using test performed on umbilical blood samples (40). The predictive value of the screening tests is not well known, while the genotyping method is a useful tool for the confirmation of G6PD diagnosis and for the family genetic screening. Parents of neonates identified as G6PD deficient should be counseled regarding the risks of jaundice linked to drugs, food intake or use of some chemicals (such as naphthalene, menthol) (41, 42) and, finally, the needing to be themselves genetically screened.

THIRD QUESTION: WHY? Some motivations suggesting the use of G6PD biochemical and molecular analyses have been above already reported extensively (Table 2). In addition, we would further highlight that, as a result of the incremental trend flow of immigration and of its changed patterns, G6PD deficiency could not be longer considered a condition limit to Southern Europe, the Middle East, and Orient. It may currently be encountered in virtually any corner of the world, also making necessary a high level of medical awareness. Some individuals are casually discovered to suffer from G6PD severe deficiency during the screening for the admission at the military school (G6PD Cassano variant): these individuals are reported to be habitual consumers of beans or users of the drugs banned for favic patients. In these cases, the complete molecular analysis is strongly indicated also considering the possible other modifiers genes potentially involved in the different clinical manifestations or phenotypes. G6PD activity dosage and genetic analysis should be also used for prevention programs, with the following objectives: (a) identification of hemizygotes males and homozygous or heterozygotes females, for the prevention of acute hemolytic crisis; (b) identification of HF, at least in families at risk, and evaluation of the neonatal jaundice of their sons at birth; (c) recognition of new mutations in G6PD deficient subjects; (d) genetic counseling for a complete family study; (e) introduction of the

GLUCOSE-6-PHOSPHATE DEHYDROGENASE LABORATORY ASSAY

Table 2 Principal recommendations for correct G6PD laboratory assay Why do the test should be made?

How do the test should be made?

When do the test should not be made?

In presence of G6PD deficiency In presence of clinical manifestations or history suggestive of G6PD-deficiency Patients belonging to a geographical area with high prevalence of G6PD deficiency Deleucocyted blood with EDTA as anticoagulant G6PD assay within 24hours after the blood collection Enzymatic quantitative assay in males G6PD/6PGD for heterozygote females or molecular test Genetic family investigation In presence of hemolytic crises In neonates, at least when possible In presence of high reticulocytes count After blood transfusion

G6PD assay in a normal protocol finalized to a biochemical check-up; (f) screening of blood donors to prevent the transfusion of deficient RBCs. On the basis of these considerations, all chemical companies should be obliged to report, on the labels or leaflets, the potential effects of drug causing hemolysis.

FURTHER STRATEGIES In the meantime we think that together with the tests commonly used for the G6PD-activity evaluation, new tests also should be developed individually for each patient. For example, an ‘‘ex-vivo’’ assay, performed on RBCs incubated with a specific toxic drug, or its metabolite, could allow to assign a rate damage in these cells in the presence of a potential oxidizing agent. This information could evaluate the influence of the individual complete compensation mechanisms giving the different phenotypes. REFERENCES 1. Sodiende, O. (1992) Glucose-6-phosphate dehydrogenase deficiency. Ballieres Clin. Haematol. 5, 367–382. 2. Mason, P. J., Bautista, J. M., and Gilsanz, F. (2007) G6PD deficiency: the genotype-phenotype association. Blood Rev. 21, 267–283. 3. Au, S. W., Naylor, C. E., Gover, S., Vandeputte-Rutten, L., Scopes, D. A., Mason, P. J., Luzzatto, L, Lam, V. M., and Adams, M. J. (1999) Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement. Acta Crystallogr. D. Biol. Crystallogr. 55, 826–384. 4. Wang, X. T., Chan, T. F., Lam, V. M., and Engel, P. C. (2008) What is the role of the second, ‘‘structural’’ NADP1 binding site in human glucose 6-phosphate dehydrogenase? Protein Sci. 17, 1403–1411.

33

5. Cappellini, M. D. and Fiorelli, G. (2008) Glucose-6-phosphate dehydrogenase deficiency. Lancet 371, 64–74. 6. Meloni, T., Forteleoni, G., Dore, A., and Cutillo, S. (1983) Favism and hemolitic anemia in glucose-6-phosphate dehydrogenase deficient subjects in North Sardinia. Acta Haematol. 70, 83–90. 7. Rigattieri, S., Silvestri, P., Minucci, A., Di Russo, C., Ferraiulo, G., Giardina, E., Capoluongo, E., and Lo Schiavo, P. (2008) Drug-eluting stents in a patient with favism: is the aspirine administration safe? J. Cardiol. Med. 9. 8. World Health Organization Working Group. (1989) Glucose-6-phosphate dehydrogenase deficiency. Bull. World Health Organ. 67, 601–611. 9. Betke, K., Brewer, G. J, Kirkman, H. N., Luzzatto, L., Motulsky, A. G., Ramot, B., Siniscalco, M. (1979) Standardization of procedure for the study of glucose-6-phosphate dehydrogenase: report of a WHO Scientific Group. World Health Org. Techn. Rep. Ser. 366, 1–53. 10. Mehta, A, Mason, P. J., and Vulliamy, T. J. (2000) Glucose-6-phosphate dehydrogenase deficiency. Baillieres Best Pract. Res. Clin. Haematol. 13, 21–38. 11. Beutler, E., Kuhl, W., Vives-Corrons, J. L., and Prchal, J. T. (1989) Molecular heterogeneity of glucose-6-phosphate dehydrogense deficiency A2. Blood 74, 2550–2555. 12. Sa´enz, G. F., Chaves, M., Berrantes, A., Elizondo, J., Montero, A. G., and Yoshida, A. (1984) A glucose-6-phosphate dehydrogenase variant, Gd(2) Santamaria found in Costa Rica. Acta Haematol. 72, 37–40. 13. Longo, L., Vanegas, O. C., Patel, M., Rosti, V., Li, H., Waka, J., Merghoub, T., Pandolfi, P. P., Notaro, R., Manova, K., and Luzzatto, L. (2002) Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal. EMBO J. 21, 4229–4239. 14. Minucci, A., Concolino, P., Vendittelli, F., Giardina, B., Zuppi, C., and Capoluongo, E. (2008) Glucose-6-phosphate dehydrogenase Buenos Aires: a novel de novo missense mutation associated with severe enzyme deficiency. Clin. Biochem. 41, 742–745. 15. Daoud, B. B., Mosbehi, I., Pre´hu, C., Chaouachi, D., Hafsia, R., and Abbes, S. (2008) Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Tunisia. Pathol. Biol. (Paris) 56, 260–267. 16. Notaro, R., Afolayan, A., and Luzzatto, L. (2000) Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history. FASEB J. 14, 485–494. 17. Tantular, I. S. and Kawamoto, F. (2003) An improved, simple screening method for detection of glucose-6-phosphate dehydrogenase deficiency. Trop. Med. Int. Health 8, 569–574. 18. Gall, J. C., Brewer, G. J., and Dern, R. J. (1965) Studies of glucose-6phosphate dehydrogenase activity of individual erythrocytes: the methemoglobin-elution test for identification of females heterozygous for G6PD deficiency. Am. J. Hum. Genet. 17, 359–368. 19. Beutler, E. (1984) Red Cell Metabolism: A Manual of Biochemical Methods, 3rd edn. Grune & Strattan, New York. 20. Oduola, T., Adeosun, G., Ogunyemi, E., Adenaike, F., and Bello, A. (2006) Studies on G6PD stability in blood stored with different anticoagulants. The Internet J. Hematol. 2. 21. Castro, S. M., Weber, R., Dadalt, V., Santos, V. F., Reclos, G. J., Pass, A., and Giugliani, R. (2005) Evaluation of glucose-6-phosphate dehydrogenase stability in blood samples under different collection and storage conditions. Clin. Chem. 51, 1080–1081. 22. Du, C., Ren, X., and Jiang, Y. (1999) Detection of three common G6PD gene point mutations in Guangdong province by using ARMS. Zhonghua Xue Ye Xue Za Zhi 20, 191–193. 23. Lam, V. M., Huang, W., Lam, S. T., Yeung, C. Y., and Johnson, P. H. (1996) Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE). Genet. Anal. 12, 201–206. 24. Zhang, D. T., Hu, L. H., and Yang, Y. Z. (2005) Detection of three common G6PD gene mutations in Chinese individuals by probe melting curves. Clin. Biochem. 38, 390–394.

34

MINUCCI ET AL.

25. Bang-Ce, Y., Hongqiong, L., and Zhensong, L. (2004) Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by microarray-based assay. Am. J. Hematol. 76, 405–412. 26. Tseng, C. P., Huang, C. L., Chong, K. Y., Hung, I. J., and Chiu, D. T. (2005) Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography. Clin. Biochem. 38, 973–980. 27. Zhao, F., Ou, X. L., Xu, C. C., Cai, G. Q., Wu, X. Y., Huang, Y. M., Zhu, W. F., and Jiang, Q. C. (2004) Rapid detection of six common Chinese G6PD mutations by MALDI-TOF MS. Blood Cells Mol. Dis. 32, 315–318. 28. Li, L., Zhou, Y. Q., Xiao, Q. Z., Yan, T. Z., and Xu, X. M. (2008) Development and evaluation of a reverse dot blot assay for the simultaneous detection of six common Chinese G6PD mutations and one polymorphism. Blood Cells Mol. Dis. 41, 17–21. 29. Farez-Vidal, M. E., Gandia-Pla, S., Blanco, S., Go´mez-Llorente, C., and Go´mez-Capilla, J. A. (2008) Multi-mutational analysis of fifteen common mutations of the glucose 6-phosphate dehydrogenase gene in the Mediterrranean population. Clin. Chim. Acta 395, 94–98 30. Minucci, A., Delibato, E., Castagnola, M., Concolino, M. Ameglio, F., Zuppi, C., Giardina, B., and Capoluongo, E. (2008) Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (ExperionTM). J. Sep. Sci. 31, 2694–2700. 31. Reclos, G. J., Hatzidakis, C. J., and Schulpis, K. H. (2000) Glucose-6phosphate dehydrogenase deficiency neonatal screening: preliminary evidence that a high percentage of partially deficient female neonates are missed during routine screening. J. Med. Screen. 7, 46–51. 32. Mosca, A., Paleari, R., Sollaino, C., Barella, S., and Galanello, R. (1998) Limits to the use of the glucose 6-phosphate dehydrogenase/6phosphogluconate dehydrogenase index for the detection of glucose 6phosphate dehydrogenase deficiency. Clin. Chem. Lab. Med. 36, 737– 738. 33. Tagarelli, A., Piro, A., Tagarelli, G., Bastone, L., Paleari, R., and Mosca, A. (2004) G6PD/PK ratio: a reliable parameter to identify glu-

34.

35.

36.

37.

38.

39.

40.

41.

42.

cose-6-phosphate dehydrogenase deficiency associated with microcytic anemia in heterozygous subjects. Clin. Biochem. 37, 863–866. Van Noorden C. J., and Vogels I. M. (1985) A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency. Br. J. Haematol. 60, 57–63. Kaplan, M. and Hammerman C. (1998) Severe neonatal hyperbilirubinemia. A potential complication of glucose-6-phosphate dehydrogenase deficiency. Clin. Perinatol. 25, 575–590. Bhutani, V. K., Johnson, L. H., Jeffrey Maisels, M., Newman, T. B., Phibbs, C., Stark, A. R., and Yeargin-Allsopp M. (2004) Kernicterus: epidemiological strategies for its prevention through systems-based approaches. J. Perinatol. 24, 650–662. Beutler, E., Blume, K. G., Kaplan, G. C., Lohr, G. W., Ramot, B., and Valentine, W. N. (1979) International comitte for standardization in haematology: recommended screening test for glucos-6-phosphate dehydrogenase (G6PD) deficiency. Br. J. Haematol. 43, 465–468. Solem, E., Pirzer, C., Siege, M., Kollmann, F., Romero-Saravia, O., Bartsch-Trefs, O., and Kornhuber, B. (1985) Mass screening for glucose-6-phosphate dehydrogenase deficiency: improved fluorescent spot test. Clin. Chim. Acta. 152, 135–142. Zaffanello, M., Rugolotto, S., Zamboni, G., Gaudino, R., and Tato`, L. (2004) Neonatal screening for glucose-6-phosphate dehydrogenase deficiency fails to detect heterozygote females. Eur. J. Epidemiol. 19, 255–257. Kaddari, F., Sawadogo, M., Sancho, J., Lelong, M., Jaby, D., Paulin, C., Nkana, K., and Cailliez, M. (2004) Neonatal screening of glucose-6phosphate dehydrogenase deficiency in umbilical cord blood. Ann. Biol. Clin. (Paris) 62, 446–450. Olowe, S. A. and Ransome-Kuti, O. (1980) The risk of jaundice in glucose-6-phosphate dehydrogenase deficient babies exposed to menthol. Acta Paediatr. Scand. 69, 341–345. Zinkham, W. H. and Oski, F. A. (1996) Henna: a potential cause of oxidative hemolysis and neonatal hyperbilirubinemia. Pediatrics 97, 707–709.