Practical approach for characterization of glucose 6‐phosphate dehydrogenase (G6PD) deficiency in countries with population ethnically heterogeneous: Description of seven new G6PD mutants
Authors: Kamran Moradkhani, Chadia Mekki, Michel Bahuau, Valerie Li Thiao Te, Muriel Holder, Serge Pissard, Claude Préhu, Christian Rose, Henri Wajcman, Frédéric Galactéros
Journal: American Journal of Hematology (2011)

We present a rapid strategy based on Restriction Fragment Length Polymorphism (RFLP) analysis to characterize the more frequent glucose 6-phosphate dehydrogenase (G6PD) variants observed in a population with high gene flow. During a study involving more than 600 patients, we observed mainly G6PD A− (c.202G>A, c.376A>G; p.Val68Met, p.Asn126Asp), G6PD Mediterranean (Med) (c.563C>T, p.Ser188Phe), and G6PD Betica (c.376A>G, 542A>T; p.126Asn>Asp, 181Asp>Val) with addition of a few rare ones. A number of 10 abnormalities amounted to 92% of all the molecular defects. In addition, seven new mutations were found: three presented with acute hemolytic anemia following oxidative stress [G6PD Nice (c.1380G>C, p.Glu460Asp), G6PD Roubaix (c.811G>C, p.Val271Leu), and G6PD Toledo (c.496C>T, p.Arg166Cys)], three with different degrees of chronic hemolytic anemia [G6PD Lille (c.821A>T, p.Glu274Val), G6PD Villeurbanne (c.1000_1002delACC, p.Thr334del), and G6PD Amiens (c.1367A>T, p.Asp456Val)] and one found fortuitously G6PD Montpellier (c.1132G>A, p.Gly378Ser). G6PD deficiency is the most frequent red blood cell (RBC) abnormality worldwide and is essentially divided into three classes [1]. Class I variants are characterized by a severe deficiency with chronic hemolytic anemia. Class II and III variants have substantial remaining enzyme activity and only present hemolytic crises under oxidant stress. [2]. Until last century, the Class II–III G6PD variants were restricted to some populations with a distribution specific to each region [3, 4]. Thus, in some countries, only very few molecular defects are encountered, while in industrialized countries, with high immigration flow, the simultaneous presence of several variants is observed. The conventional approach is a time consuming and expensive sequencing of the gene. Therefore, working in a reference laboratory where more than 100 samples are investigated yearly, we propose a simplified procedure for molecular diagnosis of this defect. During the last decade, we investigated more than 600 cases presenting a G6PD deficiency for which we could obtain an informed consent. We started this study by analyzing the entire sequence of G6PD gene. Rapidly we realized that more than 75% of abnormalities corresponded to G6PD A− and G6PD Med and that a total of 10 variants covered 92% of all the defects [5]. Therefore, we developed the herein described rapid strategy based on RFLP analysis that could easily be adapted to other countries according to the specificity of their population. First, we look for the presence of G6PD A− and Med by restriction enzymes as previously described [5]. In the remaining 25% of subjects, according to their ethnic origin, the eight other frequent abnormalities are searched for by RFLP using restriction enzymes specific for each mutation. Only the very rare remaining cases required a complete DNA analysis. In total, we found G6PD A− (62.7%), Med (11.8%), Betica (4.9%), Santa Maria (3.1%), Viangchan (3.1%), Seattle (1.8%), Canton (1.3%), Chatham (1.3%), Valladolid (1.1%), and Coimbra (0.5%). In addition, we observed some rare variants already described in the literature [6] such as G6PD Nankang in a Antillean woman, Chinese-5, in two south East Asian males, and G6PD Genova in a German woman. We observed also three cases of compound heterozygoty (G6PD Med/G6PD Seattle, G6PD Betica/G6PD Santa Maria, and G6PD A−/G6PD Santa Maria), with residual enzyme activity similar to that of hemizygotes. Seven new molecular defects were observed, corresponding DNA sequences are presented as Supporting Information. G6PD Nice (p.Glu460Asp) was found in a 35-year-old man of Italian origin admitted for a hemolytic crisis following fava bean ingestion. G6PD assay revealed 5%–10% residual enzyme activity. Family study showed that his sister and niece, who never had hemolytic crisis, presented 50% residual G6PD activity. Hematologic parameters of the niece were normal. The molecular study of G6PD locus in his sister revealed an undescribed G6PD mutation: c.1380G>C in exon 12. This residue is the one which follows that mutated in G6PD Canton. Glu 460 and Lys 293, located toward the exterior of the molecule are distant by 5 Å and forms an ionic bond (Fig. 1). In G6PD Nice, the replacement of Glu for Asp increases this distance to more than 6 Å, reducing the interaction between these two sites. Localization of the structural abnormalities in the 3D model of a G6PD tetramer. Structural modifications of G6PD Lille, Amiens, Montpellier, Roubaix, and Nice are localized near to the area involved in tetramerization. It is likely that Cys13 may form a disulfide bridge with Cys446. In G6PD Toledo, the introduction of an additional cysteine near to Cys446 could disturb the local structure presence of this region. G6PD Villeurbanne resulted from a loss of one of the two consecutive threonines at 333 and 334 may play a role in interaction of subunits forming the tetramer [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] G6PD Roubaix (Val271Leu) was found in a newborn girl presenting a Noonan syndrome and a prolonged neonatal icterus. G6PD assay revealed a deficiency, which was later on characterized at molecular level by direct sequencing. In exon 8, nucleotide 811 was replaced by a cytosine. Her mother and her sister, with normal enzymatic assay were heterozygous for the same mutation. When the proband was 2-year old, her RBC indices were normal. This mutation disturbs a hydrophobic pocket localized not very far from the interaction area between the two subunits of the active dimer (Fig. 1). G6PD Toledo (p.Arg166Cys) was found in an 11-year-old boy presenting with an acute hemolytic crisis following a viral infection. He presented a neonatal jaundice. G6PD enzyme activity was not detectable. Molecular study revealed a mutation (c.496C>T) in exon 6. No genetic abnormality was found in his mother indicating that it is a neomutation. Eight cysteines are present in the normal enzyme, and it is likely that Cys13 may form a disulfide bridge with Cys446. The introduction of a cysteine near to this region could disturb the local structure. G6PD Lille (p.Glu274Val) was found in a 23-year-old French Caucasian man. He presented, at birth, a severe jaundice requiring exchange blood transfusion. He needed further several blood transfusions until 7-year-old. He was seen again when 15-year-old for a mucocutaneous jaundice, a hyperreticulocytosis (16%). At the age of 22 years, a moderate splenomegaly and hemosiderosis were observed. The biological and hematological parameters indicated a compensated hemolysis associated to a macrocytosis (haptoglobin < 0.3 mg/l and ferritin 411 μg/l with 75% of saturation of transferrin). G6PD enzyme activity was 2 UI/g Hb. Direct DNA sequencing revealed a mutation in exon 8 of G6PD gene (c.821A>T). According to the clinical presentation, this abnormality leads to a typical G6PD Class I variant. The 3D model shows that the modified amino acid is located in the neighborhood of 275Lys, which is a key residue involved in dimerization and tetramerization of the protein (Fig. 1) [7, 8]. The same residue is affected in G6PD Cleveland (c.820G>A, p.Glu274Lys), which is another Class I variant. G6PD Villeurbanne (p.Thr334del) was found in a boy presenting a chronic hemolytic anemia and several acute hemolytic crisis following viral infections. G6PD activity is reduced (0.8 IU/g Hb). Direct sequencing of DNA revealed an in frame deletion of three nucleotides, in exon 9, resulting in the loss of one of two consecutive threonines. No molecular abnormality was found in his mother, presenting a normal G6PD activity, showing that G6PD Villeurbanne resulted from a neomutation. G6PD Amiens (p.Asp456Val) was found in a 3-year-old male with persistent scleral icterus, who had a severe neonatal jaundice. The investigations revealed a G6PD deficiency with 1% residual enzymatic activity. At the age of 6 years, his cell blood count (CBC) and biological parameters showed a chronic hemolytic anemia. When the mother was again pregnant, the G6PD deficiency was characterized. Direct sequencing of the gene revealed a yet undescribed substitution of A>T at nucleotide 1367, in exon 12 in child and her mother. This abnormality modifies the same residue as reported for G6PD Figueira da Foz (c.1366G>A, p.Asp456His) associated with a chronic hemolytic anemia [9]. In addition, the structural modification is close to that observed in G6PD Nice or Canton and may therefore affect association between subunits. G6PD Montpellier (p.Gly378Ser) was found in a 14-year-old boy of Iberian origin. During a hospitalization indicated for an abdominal trauma, a hyperbilirubinemia was fortuitously found. Hematological parameters were normal. The sonographic examination revealed the presence of gallstones. The patient was found to be homozygous for the (TA)7 allele in the promoter region of the gene for bilirubin UDP-glucuronosyltransferase-1. In addition, a complete RBC enzyme study was performed, which revealed a simultaneous presence of two enzyme defects. The first one, a pyruvate kinase (PK) deficiency with a 50% decreased activity and the second one a G6PD deficiency affecting 50% of the enzyme activity. The molecular study of PK gene revealed a common missense mutation in exon 11 (c.1456C>T) [10]. The study of G6PD gene showed a new mutation in exon 10 (c.1132G>A). This substitution affecting an amino-acid located between two β sheets could alter steric hindrance of Lys275 and may thus slightly affect association into dimers or tetramers. Human active G6PD is an equilibrium between homodimers and tetramers, while the monomer is unstable. Following its 3D structure determination, it was observed that most of the Class I variants resulted from changes of residues encoded by exon 10. These residues are located in a region that binds a molecule of NADP+ involved in the structure of the protein, thus stabilizing the contact area between subunits within the active dimer [7, 8]. A few other Type I variants alter directly the catalytic center or some key regions, well conserved during evolution, which are involved in the stability of the protein. Conversely, the Class II–III variants are found distributed all over the molecule and result in local structural changes that may affect more or less the stability or the activity of the enzyme. Among the seven new variants that we report, G6PD Montpellier is the only one encoded by a mutation in exon 10, but its amino acid modification, from a glycine to a serine, is not expected to disturb significatively the spatial structure. None of the new Class I variants here reported concerns a residue encoded by exon 10: G6PD Villeurbanne is a single residue deletion that may induce quite large local changes, G6PD Lille is near a key residue (Lys275) involved in dimerization and tetramerization of the protein, and G6PD Amiens concerns a position, whose modifications have already been described to lead to Class I variants [11]. As shown in Fig. 1, four of the new variants here reported are localized near to the contact areas between dimers and tetramers. It could therefore be hypothesized that keeping the equilibrium between dimers and tetramers is a way to prevent formation of unstable monomers. Any mutation that will impair formation of tetramers will increase the amounts of dimers and could thus displace the equilibrium favoring its dissociation. Further biochemical and biophysical studies are certainly required to validate this mechanism. This study contributes to demonstrate the heterogeneity of molecular mechanism leading to G6PD deficiency. Six hundred and four individuals with suspicion of G6PD deficiency were characterized at the molecular level. An informed consent was obtained from each patient, according to the French regulation. The majority of them originated from African and Mediterranean countries. The remaining was from Asiatic origin. In addition, some patients were sent to our laboratory for more thorough investigation because of neonatal jaundice, favism, family studies, or systematic controls before some treatments. Blood sampling was done using ethylenediaminetetraacetic acid (EDTA) as anticoagulant. Samples were shipped to the laboratory at 4°C for CBC and DNA study. Enzyme activity study was measured on ACD samples. Hematological parameters were determined by routine methods. G6PD assay was done spectrophotometrically according to Beutler et al., 1968 using a 20XTi auto-analyzer (Thermo Electron, Victoria, Australia). In a first step, we looked for G6PD A− and Med by NlaIII, FokI, and MboII restriction enzymes as previously described [5]. For the remaining cases, we look for G6PD Betica, Valladolid, and Santa Maria in individuals from North African and Mediterranean origin and G6PD Viangchan and Canton for those of Asian origin. Restriction enzymes used in this approach are listed in Table I. DNA fragments carrying these mutations were amplified using the specific primers. The list of primers is given in Supporting Information. The amplified fragments were restricted according to the manufacturer's recommendation with 20 IU of each of the corresponding enzyme. Ten microliters of the digestion products were size fractionated by electrophoresis through polyacrylamide gels (6%), stained with ethidium bromide (0.25 μg/ml), and analyzed by ultraviolet transillumination. The resulting patterns are shown in Fig. 1 of supporting materials. The creation or abolition of restriction sites is looked for. Variants detected because of abolition of a restriction site require direct sequencing for confirmation of the abnormality. Variants not detected by this approach were characterized by direct sequencing. Conditions for amplification reactions were as follows: 500 ng DNA were amplified on a GeneAmp® PCR System 2700 thermal cycler (Applied BioSystems, Foster City, CA) with 1.25 units AmpliTaq® DNA Polymerase (Roche, New Jersey, NJ) and 10 pmol of each primer, 0.2 mM each dNTP, 1.5 mM MgCl2. An initial denaturation step at 94°C for 5 min was followed by 35 cycles of denaturation at 94°C for 10 sec, annealing at 61°C for 30 sec and extension at 72°C for 30 sec. A final extension at 72°C for 7 min completed the cycling reaction. Nucleotide sequencing of the PCR products was performed with the forward and reverse primers using the BigDye® Terminator v3.1 Sequencing Kit (Applied BioSystems) and analyzed on an automated sequencer (ABI PRISM® 3100 Genetic Analyzer; Applied BioSystems). The crystallographic model of G6PD was studied using a 3D protein modeling software, Swiss pdb Viewer (http://www.expasy. ch/spdbv/text/tutorial.htm) and the pdb file 1qki [7, 8] with special attention to the affected residues. The authors acknowledge Dr Bruno Costes (CHU Henri Mondor, France) for his contribution for molecular studies. They thank Drs. Patricia Martinez (CHU Montpellier, France), Anne Lambilliotte-Lemaire (CHU Lille, France), Anne-Marie Soummer and Celine Caruba (CHU Nice, France), who sent them samples of G6PD deficient patients for more thorough molecular characterization. They also appreciate the skillful assistance of Catherine Bimet, Natacha Martin, and Claire Albert (Department of Biochemistry and Genetics, CHU Henri Mondor, Creteil, France). Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Kamran Moradkhani* , Chadia Mekki , Michel Bahuau*, Valerie Li Thiao Te , Muriel Holder§, Serge Pissard* ¶, Claude Préhu* , Christian Rose**, Henri Wajcman , Frédéric Galactéros ¶, * AP-HP, Hôpital H. Mondor-A. Chenevier, Service de Biochimie et Génétique, Créteil 94000, France, Inserm, U955, Créteil 94000, France, Service d'Oncologie-Hématologie et Immunologie Pédiatrique, CHU Amiens, 80054 Amiens, France, § Service de Génétique Clinique, Hôpital Jeanne de Flandre, CHRU Lille, 59037 Lille, France, ¶ Université Paris-Est, Faculté de Médecine, UMR955, Créteil F-94010, France, ** Service d'Hématologie, Hôpital Saint Vincent, Université Catholique de Lille, Lille, France.