African Glucose-6-Phosphate Dehydrogenase Alleles Associated with Protection from Severe Malaria in Heterozygous Females in Tanzania

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.     

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of Southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2–13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen independent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.     

G6PD Punjab,a dialysis sensitive variant of human glucose-6-phosphate dehydrogenase

Erythrocyte samples from 101 individuals, originally from Punjab and living at the time of investigation in England, were screened for glucose-6-phosphate dehydrogenase (G6PD) variants by Beutler’s fluorescent spot test and standard cellulose acetate gel (Cellogel) electrophoresis. All but 2 of the 40 males in the study were found to be indistinguishable from normal G6PD B. One of the variants had 2% of the normal activity and resembled G6PD Mediterranean in electrophoretic behaviour. The other variant showed 52% of the normal activity and migrated slower than G6PD B in Cellogel with about half of the normal band intensity. A set of physicochemical characteristics of the variant determined by conventional methods distinguished it from the variants reported so far. It was designated as G6PD Punjab, and the corresponding allele asG6PD PUN. The most striking feature of G6PD Punjab is a remarkable alteration in its electrophoretic behaviour after dialysis.     

Characterization of glucose-6-phosphate dehydrogenase deficiency and identification of a novel haplotype 487G>A/IVS5-612(G>C) in the Achang population of southwestern China

The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency and its gene mutations were studied in the Achang population from Lianghe County in Southwestern China. We found that 7.31% (19 of 260) males and 4.35% (10 of 230) females had G6PD deficiency. The molecular analysis of G6PD gene exons 2―13 was performed by a PCR-DHPLC-Sequencing or PCR-Sequencing. Sixteen inde-pendent subjects with G6PD Mahidol (487G>A) and the new polymorphism IVS5-612 (G>C), which combined into a novel haplotype, were identified accounting for 84.2% (16/19). And 100% Achang G6PD Mahidol were linked to the IVS5-612 C. The percentage of G6PD Mahidol in the Achang group is close to that in the Myanmar population (91.3% 73/80), which implies that there are some gene flows between Achang and Myanmar populations. Interestingly, G6PD Canton (1376G>T) and G6PD Kaiping (1388G>A), which were the most common G6PD variants from other ethnic groups in China, were not found in this Achang group, suggesting that there are different G6PD mutation profiles in the Achang group and other ethnic groups in China. Our findings appear to be the first documented report on the G6PD genetics of the AChang people, which will provide important clues to the Achang ethnic group origin and will help prevention and treatment of malaria in this area.     

A new variant of human erythrocyte G6PD occurring at a high frequency amongst the population of two villages in The Gambia,West Africa

Summary During the course of a large survey of red cell G6PD genotypes in The Gambia, a slow electrophoretic variant with reduced enzyme activity was found to occur at a high frequency. This variant, G6PD Gambia, was found in the following genotypic combinations: males; G6PD^Gam, females; G6PD^A+/Gam, G6PD^B+/Gam, and G6PD^A-/Gam. From the electrophoretic mobility and kinetic characteristics it was concluded that G6PD Gambia was a hitherto unreported variant of G6PD. The frequency of the G6PD^Gam gene amongst the 1109 individuals examined was 0.024.     

G6PD deficient alleles and haplotype analysis of human G6PD locus in São Tomé e Príncipe (West Africa)

A population sample from S?o Tomé e Príncipe (West Africa) was screened for the G6PD-deficient variants A- (376G/202A), Betica (376G/968C), and Santa Maria (376G/542T). G6PD locus haplotype diversity was also investigated using six intragenic RFLPs (FokI, PvuII, BspHI, PstI, BclI, NlaIII) and a (CTT)n microsatellite 18.61 kb within the G6PD locus. The estimated frequencies of the G6PD*B normal allele, the G6PD*A variant (376G), and the G6PD*A- allele were 0.698, 0.194, and 0.108, respectively. G6PD variants Betica and Santa Maria were not found. Similar levels of microsatellite diversity were found on variants G6PD*B and G6PD*A (H = 0.61 and 0.68, respectively), indicating a similar age for both alleles. All G6PD*A- alleles share the RFLP-microsatellite haplotype ++(-)+(-)+/195, the same haplotype described in nearly all the *A-alleles from sub-Saharan, Mexican Mestizo, and Portuguese populations, consistent with a single and recent origin of the G202A mutation on this *A haplotype.     

A novel point mutation in a class IV glucose-6-phosphate dehydrogenase variant (G6PD São Paulo) and polymorphic G6PD variants in São Paulo State, Brazil

In this study, we used red cell glucose-6-phosphate dehydrogenase (G6PD) activity to screen for G6PD-deficient individuals in 373 unrelated asymptomatic adult men who were working with insecticides (organophosphorus and carbamate) in dengue prevention programs in 27 cities in São Paulo State, Brazil. Twenty-one unrelated male children suspected of having erythroenzymopathy who were attended at hospitals in São Paulo city were also studied. Fifteen of the 373 adults and 12 of the 21 children were G6PD deficient. G6PD gene mutations were investigated in these G6PD-deficient individuals by using PCR-RFLP, PCR-SSCP analysis and DNA sequencing. Twelve G6PD A-202A/376G and two G6PD Seattle844C, as well as a new variant identified as G6PD São Paulo, were detected among adults, and 11 G6PD A-202A/376G and one G6PD Seattle844C were found among children. The novel mutation c.660C > G caused the replacement of isoleucine by methionine (I220M) in a region near the dimer interface of the molecule. The conservative nature of this mutation (substitution of a nonpolar aliphatic amino acid for another one) could explain why there was no corresponding change in the loss of G6PD activity (64.5% of normal activity in both cases).     

Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP⁺-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP⁺ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP⁺ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PD^def). We first solved the crystal structure for G6PD^K403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP⁺ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PD^def), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PD^K403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PD^def and provide a foundation for future drug discovery efforts.     

Erythrocyte glucose-6-phosphate dehydrogenase in the Niokolonko (Malinke of the Niokolo) of Eastern Senegal. Identification of a slow variant with normal activity (Tacoma-like).

In a survey of blood genetic markers in the Niokolonko of Eastern Senegal, three types of G6PD variants were discovered: (1) fast variants, common Negro G6PD A +; the frequency of the Gd A + gene was 0.183; (2) deficient G6PD A--, occurring with a fairly low frequency: 0.079, and (3) some individuals were carriers of a slow moving electrophoretic variant with normal activity. After purification, the analysis of kinetic parameters showed that this enzyme was closely similar to G6PD Tacoma. We proposed to label it 'G6PD Tacoma-like'. The incidence of this mutation in the whole group studied was 0.020. G6PD Tacoma-like may be common in some African tribes.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER

From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10^-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.     

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on a population from the Matera district

Summary Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A^–): three new polymorphic variants. G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic-area and they prove the presence in the Italian peninsula of a sene (Gd ^A–) regarded as characteristically African.     

Identifying Darwinian Selection Acting on Different Human APOL1 Variants among Diverse African Populations

Disease susceptibility can arise as a consequence of adaptation to infectious disease. Recent findings have suggested that higher rates of chronic kidney disease (CKD) in individuals with recent African ancestry might be attributed to two risk alleles (G1 and G2) at the serum-resistance-associated (SRA)-interacting-domain-encoding region of APOL1. These two alleles appear to have arisen adaptively, possibly as a result of their protective effects against human African trypanosomiasis (HAT), or African sleeping sickness. In order to explore the distribution of potential functional variation at APOL1, we studied nucleotide variation in 187 individuals across ten geographically and genetically diverse African ethnic groups with exposure to two Trypanosoma brucei subspecies that cause HAT. We observed unusually high levels of nonsynonymous polymorphism in the regions encoding the functional domains that are required for lysing parasites. Whereas allele frequencies of G2 were similar across all populations (3%–8%), the G1 allele was only common in the Yoruba (39%). Additionally, we identified a haplotype (termed G3) that contains a nonsynonymous change at the membrane-addressing-domain-encoding region of APOL1 and is present in all populations except for the Yoruba. Analyses of long-range patterns of linkage disequilibrium indicate evidence of recent selection acting on the G3 haplotype in Fulani from Cameroon. Our results indicate that the G1 and G2 variants in APOL1 are geographically restricted and that there might be other functional variants that could play a role in HAT resistance and CKD risk in African populations.     

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD-deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A^?202A/376G , G6PD A^?376G/968C and G6PD Santamaria^376G/542T . Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF^193A>G (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.     

Development of allelic discrimination assay to detect Mediterranean G6PD mutation and its linked inheritance with normal vision and/colorblindness loci for 4 generations among Egyptian and Emirati families

G6PD deficiency c563T is the most common inherent blood disease among the Mediterranean populations and its molecular diagnosis is critical as the enzyme assay fails for heterozygous individuals. The purpose of the study is to estimate the ubiquity of the heterozygous G6PD Med (c563T) variants among Egyptians and UAE nationals living in Dubai. We validated two molecular methods, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and qPCR allelic discrimination assay for detection of G6PD Med variants. Among 100 screened individuals, G6PD c563T variants are 30% of whom 15% are carriers. Sanger sequencing validated the qPCR discrimination assays. In search of a phenotypic marker to detect G6PD heterozygous variants, inheritance of G6PD locus and red-green color vision genes is studied in 1 Egyptian and 2 Emirati families. Among the 3 families, G6PD is polymorphic, displaying 4 phenotypes: in phenotype-1, person is normal, in phenotype-2 the person has no G6PD deficiency but with deuteranopia/deuteranomaly, in phenotype-3 the person is G6PD Med variant with deuteranopia/deuteranomaly and in phenotype 4 the person is G6PD Med variant has normal vision. Based on the molecular analysis of G6PD and Ishihara vision test it can be concluded that the two mutations at the two loci arose independent of each other without any interaction (epistatic effect) between them. Following the pedigree analysis of the two genes for 4 generations it is presumed that it is infeasible to use “deuteranopia /deuteranomaly” as a phenotypic marker to detect G6PD c563T heterozygous individuals among the Egyptian populations.     

Exonic Re-Sequencing of the Chromosome 2q24.3 Parkinson’s Disease Locus

Genome-wide association studies (GWAS) in Parkinson’s disease (PD) have identified over 20 genomic regions associated with disease risk. Many of these loci include several candidate genes making it difficult to pinpoint the causal gene. The locus on chromosome 2q24.3 encompasses three genes: B3GALT1, STK39, and CERS6. In order to identify if the causal variants are simple missense changes, we sequenced all 31 exons of these three genes in 187 patients with PD. We identified 13 exonic variants including four non-synonymous and three insertion/deletion variants (indels). These non-synonymous variants and rs2102808, the GWAS tag SNP, were genotyped in three independent series consisting of a total of 1976 patients and 1596 controls. Our results show that the seven identified 2q24.3 coding variants are not independently responsible for the GWAS association signal at the locus; however, there is a haplotype, which contains both rs2102808 and a STK39 exon 1 6bp indel variant, that is significantly associated with PD risk (Odds Ratio [OR] = 1.35, 95% CI: 1.11–1.64, P = 0.003). This haplotype is more associated than each of the two variants independently (OR = 1.23, P = 0.005 and 1.10, P = 0.10, respectively). Our findings suggest that the risk variant is likely located in a non-coding region. Additional sequencing of the locus including promoter and regulatory regions will be needed to pinpoint the association at this locus that leads to an increased risk to PD.     

Prevalence and Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency at the China-Myanmar Border

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary disease that predisposes red blood cells to oxidative damage. G6PD deficiency is particularly prevalent in historically malaria-endemic areas. Use of primaquine for malaria treatment may result in severe hemolysis in G6PD deficient patients. In this study, we systematically evaluated the prevalence of G6PD deficiency in the Kachin (Jingpo) ethnic group along the China-Myanmar border and determined the underlying G6PD genotypes. We surveyed G6PD deficiency in 1770 adult individuals (671 males and 1099 females) of the Kachin ethnicity using a G6PD fluorescent spot test. The overall prevalence of G6PD deficiency in the study population was 29.6% (523/1770), among which 27.9% and 30.6% were males and females, respectively. From these G6PD deficient samples, 198 unrelated individuals (147 females and 51 males) were selected for genotyping at 11 known G6PD single nucleotide polymorphisms (SNPs) in Southeast Asia (ten in exons and one in intron 11) using a multiplex SNaPshot assay. Mutations with known association to a deficient phenotype were detected in 43.9% (87/198) of cases, intronic and synonymous mutations were detected alone in 34.8% (69/198) cases and no mutation were found in 21.2% (42/198) cases. Five non-synonymous mutations, Mahidol 487G>A, Kaiping 1388G>A, Canton 1376G>T, Chinese 4 392G>T, and Viangchan 871G>A were detected. Of the 87 cases with known deficient mutations, the Mahidol variant was the most common (89.7%; 78/87), followed by the Kaiping (8.0%; 7/87) and the Viangchan (2.2%; 2/87) variants. The Canton and Chinese 4 variants were found in 1.1% of these 87 cases. Among them, two females carried the Mahidol/Viangchan and Mahidol/Kaiping double mutations, respectively. Interestingly, the silent SNPs 1311C>T and IVS11nt93T>C both occurred in the same 95 subjects with frequencies at 56.4% and 23.5% in tested females and males, respectively (P<0.05). It is noteworthy that 24 subjects carrying the Mahidol mutation and two carrying the Kaiping mutation also carried the 1311C>T/IVS11nt93T>C SNPs. Further studies are needed to determine the enzyme levels of the G6PD deficient people and presence of additional G6PD mutations in the study population.