Molecular heterogeneity underlying the G6PD Mediterranean phenotype

Summary As part of a study aiming to define the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency, we analysed a sample from a Portugese boy with a family history of favism. Although the biochemical properties of red-cell G6PD from this subject were similar to those of the common variant G6PD Mediterranean, the corresponding mutation (563 CT) was not present. Instead, polymerase chain reaction (PCR) amplification and sequencing of the entire gene detected a CT transition at nucleotide 592 in exon VI, changing an arginine residue to a cysteine residue only 10 amino acids downstream from the Mediterranean mutation. Single-strand conformation polymorphism analysis of a PCR-amplified DNA fragment spanning exons VI and VII of the G6PD gene has detected the same mutation, confirmed by sequencing, in a G6PD-deficient patient from Southern Italy. We name this new variant G6PD Coimbra.     

Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu archipelago (southwestern Pacific).

In studying the relationship between genetic abnormalities of red blood cells and malaria endemicity in the Vanuatu archipelago in the southwestern Pacific, we have found that of 1,442 males tested, 98 (6.8%) were G6PD deficient. The prevalence of GdPD deficiency varied widely (0%-39%), both from one island to another and in different parts of the same island, and generally correlated positively with the degree of malaria transmission. The properties of G6PD from GdPD-deficient subjects were analyzed in a subset of 53 samples. In all cases the residual red-blood-cell activity was < 10%. There were three phenotypic patterns. PCR amplification and sequencing of the entire coding region of the G6PD gene showed that the first of these patterns corresponded to G6PD Union (nucleotide 1360C-->T; amino acid 454Arg-->Cys), previously encountered elsewhere. Analysis of samples exhibiting the second pattern revealed two new mutants: G6PD Vanua Lava (nucleotide 383T-->C; amino acid 128Leu-->Pro) and G6PD Namoru (nucleotide 208T-->C; amino acid 70Tyr-->His); in three samples, the underlying mutation has not yet been identified. Analysis of the sample exhibiting the third pattern revealed another new mutant: G6PD Naone (nucleotide 497G-->A; amino acid 166Arg-->His). Of the four mutations, G6PD Union and G6PD Vanua Lava have a polymorphic frequency in more than one island; and G6PD Vanua Lava has also been detected in a sample from Papua New Guinea. G6PD deficiency is of clinical importance in Vanuatu because it is a cause of neonatal jaundice and is responsible for numerous episodes of drug-induced acute hemolytic anemia.     

Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system.

Glucose-6-phosphate dehydrogenase (G6PD) is the most common human enzymopathy. To date more than 122 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. The 2 most common mutations, G1388A and G1376T, account for more than 50% of mutations representing various regions and ethnic groups in China. Setting up a simple and accurate method for detecting these mutations is not only useful for studying the frequency of the G6PD genotypes, but also for finding new mutations. The purpose of this study was to find a simple, inexpensive and accurate method for detecting these common mutations. The amplification refractory mutation system (ARMS) method was used in this study. Samples from 28 G6PD-deficient males were investigated. The natural and mismatched amplification and restriction enzyme digestion method was used as a standard method to evaluate the nature of the point mutations. Sixteen cases were found carrying the G1388A mutation and 12 the G1376T mutation. Fourteen cases of G1388A and 10 cases of G1376T were confirmed by ARMS. Four cases were not in concordance with the results obtained by the mismatched amplification-restriction enzyme digestion. These 4 cases were then judged by direct PCR sequencing at exon 12. The DNA sequencing data supported the results obtained by ARMS. Thus we concluded that the ARMS is a rapid, simple, inexpensive and accurate method for detecting the most common G6PD gene mutations among the Chinese.     

First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).

Methods

Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.

Results

Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.

Conclusions

No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.     

Origin and spread of the glucose-6-phosphate dehydrogenase variant (G6PD-Mediterranean) in the Middle East.

A common glucose-6-phosphate dehydrogenase (G6PD) variant characterized by severe enzyme deficiency and B-like electrophoretic mobility is called "G6PD-Mediterranean" because it is found in different populations around the Mediterranean Sea. Sequence analysis of Italian subjects has revealed that the molecular basis of G6PD-Mediterranean is a single C-T transition at nucleotide position 563, causing a serine phenylalanine replacement at amino acid position 188. Most G6PD-Mediterranean subjects also have a silent C-T transition (without amino acid replacement) at nucleotide position 1311. Twenty-one unrelated individuals from Saudi Arabia, Iraq, Iran, Jordan, Lebanon, and Israel with both severe G6PD deficiency and B-like electrophoretic mobility were tested for both mutations by using amplification followed by digestion with appropriate restriction enzymes. All but one had the 563 mutation, and, of these, all but one had the 1311 mutation. Another 24 unrelated Middle Eastern individuals with normal G6PD activity or not known to be G6PD deficient were similarly tested. Four had the silent mutation at position 1311 in the absence of the deficiency mutation at position 563. We conclude that (1) the large majority of Middle Eastern subjects with the G6PD-Mediterranean phenotype have the same mutation found in Italy, (2) the silent mutation is an independent polymorphism in the Middle East, with a frequency of about .13, and (3) the mutation leading to the G6PD-Mediterranean deficiency has probably arisen on a chromosome that already carried the silent mutation.     

G6PD Ferrara I has the same two mutations as G6PD A(-) but a distinct biochemical phenotype

The cloning and sequencing of the normal glucose-6-phosphate dehydrogenase (G6PD) gene has led to the study of the molecular defects that determine enzymatic variants. In this paper, we describe the mutations responsible for the Ferrara I variant in an Italian man with a family history of favism, from the Po delta. Nucleotide sequencing of this variant showed a GA mutation at nucleotide 202 in exon IV causing a ValMet amino acid exchange, and a second AG mutation at nucleotide 376 in exon V causing an AsnAsp amino acid substitution. Although on the basis of its biochemical properties this variant was classified as G6PD Ferrara I, it has the same two mutations as G6PD A(-), which is common in American and African blacks, and as the sporadic Italian G6PD Matera. The mutation at nucleotide 202 was confirmed by NlaIII digestion of a polymerase chain reaction amplified DNA fragment spanning 109 bp of exon IV. The 109-bp mutated amplified sequence is not distinguishable from the normal sequence in single strand conformation polymorphism analysis.     

Molecular genetics of the glucose-6-phosphate dehydrogenase (G6PD) Mediterranean variant and description of a new G6PD mutant, G6PD Andalus1361A

Glucose-6-phosphate dehydrogenase (G6PD; E.C.1.1.1.49) deficiency is the most common human enzymopathy; more than 300 different biochemical variants of the enzyme have been described. In many parts of the world the Mediterranean type of G6PD deficiency is prevalent. However, G6PD Mediterranean has come to be regarded as a generic term applied to similar G6PD mutations thought, however, to represent a somewhat heterogeneous group. A C----T mutation at nucleotide 563 of G6PD Mediterranean has been identified by Vulliamy et al., and the same mutation has been found by De Vita et al. in G6PD Mediterranean, G6PD Sassari, and G6PD Cagliari. The latter subjects had an additional mutation, at nucleotide 1311, that did not produce a coding change. We have examined genomic DNA of five patients--four of Spanish origin and one of Jewish origin--having enzymatically documented G6PD Mediterranean. All had both the mutation at nucleotide 563 and that at nucleotide 1311. A sixth sample, resembling G6PD Mediterranean kinetically but with a slightly rapid electrophoretic mobility, was designated G6PD Andalus and was found to have a different mutation, a G----A transition at nucleotide 1361, producing an arginine-to-histidine substitution. These studies suggest that G6PD Mediterranean is, after all, relatively homogeneous.     

Molecular abnormality of G6PD Konan and G6PD Ube,the most common glucose-6-phosphate dehydrogenase variants in Japan

G6PD Konan and G6PD Ube are the most common glucose-6-phosphate dehydrogenase (G6PD) variants found in Japan. To clarify the molecular abnormality of these two variants, the entire coding region was amplified by polymerase chain reaction from genomic DNA (G6PD Konan) or cDNA (G6PD Ube). Direct sequencing revealed that both variants have the same nucleotide substitution (241 C to T) in exon 4, which predicts an Arg to Cys substitution at amino acid 81.     

Investigation of the Mutation Points and Effects of Some Drugs on Glucose-6-phosphate Dehydrogenase-deficient People in the Erzurum Region

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on three samples of 1,183 children aged 0.5–6 years from Erzurum, in eastern Anatolia. Total genomic DNAs were isolated from the blood samples of a healthy person and the three persons determined with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Then PCR amplification of the entire coding region in eight fragments was carried out followed by Agarose gel electrophoresis. The 540-bp PCR fragment containing exons VI-VII and the 550 bp PCR fragment containing exons XI-XIII were digested with EcoRI and with NIaIII, respectively. SSCP techniques for eight fragments (exons II, III-IV, V, VI-VII, VIII, IX, X, and XI-XIII) were employed to determine the mutations on the exons of the G6PD gene. A mutation occurred on the region of the exons 6 and 7 of one person (person-1) and exon 5 of two G6PD-deficient persons (person 2 and 3) examined. The sequential approach described is fast and efficient and could be applied to other populations.

Effects of analgesic drugs on G6PD were studied on the purified enzyme (ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography) for the healthy person and G6PD-deficient persons 1, 2 and 3. The effects of remifentanil hydrochloride, fentanyl citrate, alfentanil hydrochloride and pethidine hydrochloride, as analgesic drugs, on G6PD activity were tested. Although remifentanil hydrochloride, fentanyl citrate (I₅₀ values; 1.45 mM and 6.1 mM, respectively) inhibited the activity of the enzyme belonging to the healthy person, they did not alter enzyme activity on two of the three persons with G6PD deficiency. Other drugs (alfentanil hydrochloride and pethidine hydrochloride) did not effect the enzyme activity of the healthy or G6PD-deficient children.     

Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.     

Neonatal screening of G6PD deficiency in Tunisia

The Glucose-6-phosphate dehydrogenase (G6PI) deficiency is the most common enzymopathy worldwide. WHO had classified Tunisia among countries that are moderately affected by this affection. However, no mass-screening reflecting the real incidence was realized. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis in Tunisia. A total of 1102 neonates, born in CMNT center of Maternity and of Neonatology of Tunis during the going periods from April, 2005 till May, 2005 and from June, 2006 till September, 2006, have been enclosed in the study. The samplings included 953peripheral venous blood and 149 blood cordon. Among 1102 samplings, only 976 were of use to the screening. In our mass-screening, we consider all newborns that were born in the CMNT during the period of study and were included in the screening. A dosage of the enzymatic activity was realized using spectrophotometric method. G6PD electrophoresis and molecular study by PCR/RFLP were realized for the overdrawn newborn children. Among 976 screening neonates, 43 individuals (4.4%) were found to be G6PD deficient by quantitative enzyme assay. Newborn affected were distributed in 23 boys and 20 girls (sex ratio of 1.15). The electrophoretic mobility and the molecular biology were realized for the affected newborn. Molecular characterization of 30 G6PD deficient neonates revealed that the G6PD A- was the most common and was detected in 20 of 43 individuals (66.7%), followed by G6PD Mediterranean that was detected in 6 (13.3%). At least, 4 other unknown mutations were not able to be determined by PCR/RFLP (n=4). In conclusion G6PD deficiency is frequent in our country, justifying a systematic neonatal screening, to avoid the arisen of grave consequences of this affection. The African variant is the most frequent in our country followed by the Mediterranean one.     

Chronic nonspherocytic hemolytic anemia (CNSHA) and glucose 6 phosphate dehydrogenase (G6PD) deficiency in a patient with familial amyloidotic polyneuropathy (FAP)

Summary A new glucose-6-phosphate dehydrogenase (G6PD) variant with severe erythrocytic G6PD deficiency and a unique pH optimum is described in a young patient with chronic nonspherocytic hemolytic anemia (CNSHA) and familial amyloidotic polyneuropathy (FAP). Chronic hemolysis was present in the absence of infections, oxidant drugs or ingestion of faba beans. Residual enzyme activity was about 2.6% and 63% of normal activity in erythrocytes and leucocytes, respectively. A molecular study using standard methods showed G6PD in the patient to have normal electrophoretic mobility (at pH 7.0, 8.0 and 8.8), normal apparent affinity for substrates (K_m, G6P and NADP) and a slightly abnormal utilization of substrate analogues (decreased deamino-NADP and increased 2-deoxyglucose-6-phosphate utilization). Heat stability was found to be markedly decreased (8% of residual activity after 20 min of incubation at 46°C) and a particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH. Although molecular characteristics of this variant closely resemble those of G6PD Bangkok and G6PD Duarte, it can be distinguished from these and all other previously reported variants by virtue of its unusual pH curve. Therefore the present variant has been designated G6PD Clinic to distinguish it from other G6PD variants previously described.     

Investigation of the mutation points and effects of some drugs on glucose-6-phosphate dehydrogenase-deficient people in the Erzurum region

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on three samples of 1,183 children aged 0.5-6 years from Erzurum, in eastern Anatolia. Total genomic DNAs were isolated from the blood samples of a healthy person and the three persons determined with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Then PCR amplification of the entire coding region in eight fragments was carried out followed by Agarose gel electrophoresis. The 540-bp PCR fragment containing exons VI-VII and the 550bp PCR fragment containing exons XI-XIII were digested with EcoRI and with NIaIII, respectively. SSCP techniques for eight fragments (exons II, III-IV, V, VI-VII, VIII, IX, X, and XI-XIII) were employed to determine the mutations on the exons of the G6PD gene. A mutation occurred on the region of the exons 6 and 7 of one person (person-1) and exon 5 of two G6PD-deficient persons (person 2 and 3) examined. The sequential approach described is fast and efficient and could be applied to other populations. Effects of analgesic drugs on G6PD were studied on the purified enzyme (ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography) for the healthy person and G6PD-deficient persons 1, 2 and 3. The effects of remifentanil hydrochloride, fentanyl citrate, alfentanil hydrochloride and pethidine hydrochloride, as analgesic drugs, on G6PD activity were tested. Although remifentanil hydrochloride, fentanyl citrate (I50 values; 1.45mM and 6.1 mM, respectively) inhibited the activity of the enzyme belonging to the healthy person, they did not alter enzyme activity on two of the three persons with G6PD deficiency. Other drugs (alfentanil hydrochloride and pethidine hydrochloride) did not effect the enzyme activity of the healthy or G6PD-deficient children.     

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.     

Glucose-6-phosphate dehydrogenase (G6PD) Iserlohn and G6PD Regensburg: two new severe enzyme defects in German families

Two new inheritable variants of glucose-6-phosphate dehydrogenase have been found in two unrelated German families. Patients with one variant (G6PD Iserlohn, also referred to as G6PD I) suffered from intermittent hemolytic crises caused by fava beans; patients with the other variant (G6PD Regensburg, G6PD II) disclosed chronic nonspherocytic hemolytic anemia aggravated by drug treatment. Due to their unusual biochemical characteristics, the new variants were designated G6PD Iserlohn and G6PD Regensburg. Both variants showed a reduction of enzyme activity to about 6% of the normal in erythrocytes, normal electrophoretic mobility, increased affinity for glucose-6-phosphate, a reduced affinity for NADP and a pH optimum in the neutral region (7.0 and 7.5). G6PD Iserlohn had a decreased affinity for the inhibitor NADPH; G6PD Regensburg had a normal inhibitor constant. Deamino NADP was utilized at an increased rate by G6PD Regensburg. G6PD Iserlohn was thermostable, G6PD Regensburg mildly instable. G6PD activity in leukocytes was normal in G6PD Iserlohn and reduced to the same degree as in erythrocytets in G6PD Regensburg. The cause of the decreased activity of G6PD Iserlohn appears to be in vivo instability; in G6PD Regensburg further mechanisms might include reduced specific activity or reduced synthesis of the variant enzyme.     

G6PD Viangchan: a new glucose 6-phosphate dehydrogenase variant from Laos

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase-(G6PD) variant. G6PD Viangchan was found in a Laotian immigrant to Calgary, Canada, and was characterized by severe enzyme deficiency, normal electrophoretic mobility, increased pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP, as well as the artificial substrates 2-deoxy G6PD and deamino NADP. The inhibition constant for NADPH was decreased. The subject has no evidence suggesting chronic or episodic hemolysis.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

G6PD lozere and trinacria-like

Summary Two new G6PD variants have been found in red blood cells of the members of a French family originating from Lozere. The father is hemizygous for an electrophoretically fast variant with mild enzyme deficiency (50–60% of normal). The abnormal paternal G6PD gene is segregating in his daughter who is double heterozygous for maternal and paternal variants. This mutant enzyme, different from previously described variants is designated as Gd Lozère. The mother is heterozygous for another G6PD variant. Two sons are hemizygous for this latter mutant enzyme characterized by a moderate deficiency (25–30% of normal) and slower electrophoretic mobility with some slightly altered kinetic properties. This G6PD has been identified as Gd Trinacria like.These two abnormal enzymes are not associated with any hemolytic problem. Case reported is the first showing the segregation of two new mutant enzymes, distinct from common G6PD variants, among the members of the same family.     

A New Glucose-6-Phosphate Dehydrogenase Variant, G6PD Orissa (44 Ala→Gly), is the Major Polymorphic Variant in Tribal Populations in India

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been endemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala→Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser→Phe) variant. The K of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site.     

Large Cohort Screening of G6PD Deficiency and the Mutational Spectrum in the Dongguan District in Southern China

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymatic disorder of the erythrocytes that affects 400 million people worldwide. We developed a PCR-reverse dot blot (RDB) assay to screen twenty genotypes of seventeen Chinese G6PD mutations and investigate the spectrum of G6PD deficiency mutations in Dongguan District, Guangdong Province, in southern China.

Method

The PCR-RDB assay consists of multiplex PCR amplification of seven fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. A total of 16,464 individuals were analyzed by a combination of phenotypic screening and genotypic detection using the PCR-RDB assay and DNA sequence analysis.

Results

The PCR-RDB assay had a detection rate of 98.1%, which was validated by direct sequencing in a blind study with 100% concordance. The G6PD deficiency incidence rate in Dongguan District is 4.08%. Thirty-two genotypes from 469 individuals were found. The two most common variants were c.1376G>T and c.1388G>A, followed by c.95A>G, c.871G>A, c.392G>T, and c.1024 C>T. In addition, two rare mutations (c.703C>A and c.406C>T) were detected by DNA sequencing analysis. In our study, 65 cases harbored the C1311T/IVS polymorphism and 67 cases were homozygote.

Conclusion

The PCR-RDB assay we established is a reliable and effective method for screening G6PD mutations in the Chinese population. Data on the spectrum of mutations in the Dongguan District is beneficial to the clinical diagnosis and prevention of G6PD deficiency.