G6PD Locus Research Articles

PCR based method for sex estimation from bone samples of unidentified South African fetal remains

Fragmentary bone in disaster victim identification is reliant on a robust and objective method. Current methods employing traditional phenotypic analysis for sex estimation of forensic fetal remains are limited and often not possible due to the lack of distinctive cranial and pelvic traits. Thus, a molecular sex estimation method optimized for bone samples would enhance current techniques. Sternal rib samples were dissected from unclaimed perinatal decedents, presenting at varied stages of decomposition and unconfirmed circumstances of death, who were admitted to the Johannesburg Forensic Pathology Service (JFPS) and included in the Johannesburg Forensic Paediatric Collection (JFPC). A molecular protocol was designed and optimized using genomic DNA from SW480 cells and five individuals of unknown provenance and known sex (estimated during medicolegal autopsy) by targeting the X-linked G6PD and Y-linked SRY genes. The sex estimated at autopsy was confirmed using the optimized protocol. Nine (9) unidentified individuals were included and assigned sex based on the method described. The development of a sex typing protocol targeting the G6PD and SRY loci provides a new PCR method for sex estimation of human remains from 25 mg of bone fragment.

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.

Risk of pneumococcal bacteremia in Kenyan children with glucose-6-phosphate dehydrogenase deficiency

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency state in humans. The clinical phenotype is variable and includes asymptomatic individuals, episodic hemolysis induced by oxidative stress, and chronic hemolysis. G6PD deficiency is common in malaria-endemic regions, an observation hypothesized to be due to balancing selection at the G6PD locus driven by malaria. G6PD deficiency increases risk of severe malarial anemia, a key determinant of invasive bacterial disease in malaria-endemic settings. The pneumococcus is a leading cause of invasive bacterial infection and death in African children. The effect of G6PD deficiency on risk of pneumococcal disease is undefined. We hypothesized that G6PD deficiency increases pneumococcal disease risk and that this effect is dependent upon malaria.MethodsWe performed a genetic case-control study of pneumococcal bacteremia in Kenyan children stratified across a period of falling malaria transmission between 1998 and 2010.ResultsFour hundred twenty-nine Kenyan children with pneumococcal bacteremia and 2677 control children were included in the study. Among control children, G6PD deficiency, secondary to the rs1050828 G>A mutation, was common, with 11.2% (n = 301 of 2677) being hemi- or homozygotes and 33.3% (n = 442 of 1329) of girls being heterozygotes. We found that G6PD deficiency increased the risk of pneumococcal bacteremia, but only during a period of high malaria transmission (P = 0.014; OR 2.33, 95% CI 1.19–4.57). We estimate that the population attributable fraction of G6PD deficiency on risk of pneumococcal bacteremia in areas under high malaria transmission is 0.129.ConclusionsOur data demonstrate that G6PD deficiency increases risk of pneumococcal bacteremia in a manner dependent on malaria. At the population level, the impact of G6PD deficiency on invasive pneumococcal disease risk in malaria-endemic regions is substantial. Our study highlights the infection-associated morbidity and mortality conferred by G6PD deficiency in malaria-endemic settings and adds to our understanding of the potential indirect health benefits of improved malaria control.

Reappraisal of known malaria resistance loci in a large multicenter study.

Many human genetic associations with resistance to malaria have been reported but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. There was strong evidence of association with the HBB, ABO, ATP2B4, G6PD and CD40LG loci but previously reported associations at 22 other loci did not replicate in the multi-centre analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anaemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

Genome-wide association analysis of red blood cell traits in African Americans: the COGENT Network.

Laboratory red blood cell (RBC) measurements are clinically important, heritable and differ among ethnic groups. To identify genetic variants that contribute to RBC phenotypes in African Americans (AAs), we conducted a genome-wide association study in up to ~16 500 AAs. The alpha-globin locus on chromosome 16pter [lead SNP rs13335629 in ITFG3 gene; P < 1E-13 for hemoglobin (Hgb), RBC count, mean corpuscular volume (MCV), MCH and MCHC] and the G6PD locus on Xq28 [lead SNP rs1050828; P < 1E - 13 for Hgb, hematocrit (Hct), MCV, RBC count and red cell distribution width (RDW)] were each associated with multiple RBC traits. At the alpha-globin region, both the common African 3.7 kb deletion and common single nucleotide polymorphisms (SNPs) appear to contribute independently to RBC phenotypes among AAs. In the 2p21 region, we identified a novel variant of PRKCE distinctly associated with Hct in AAs. In a genome-wide admixture mapping scan, local European ancestry at the 6p22 region containing HFE and LRRC16A was associated with higher Hgb. LRRC16A has been previously associated with the platelet count and mean platelet volume in AAs, but not with Hgb. Finally, we extended to AAs the findings of association of erythrocyte traits with several loci previously reported in Europeans and/or Asians, including CD164 and HBS1L-MYB. In summary, this large-scale genome-wide analysis in AAs has extended the importance of several RBC-associated genetic loci to AAs and identified allelic heterogeneity and pleiotropy at several previously known genetic loci associated with blood cell traits in AAs.

G6PD Deficient Alleles and Haplotype Analysis of Human G6PD Locus in São Tomé e Príncipe (West Africa)

A population sample from São 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.

Genetic isolation between Atlantic and Mediterranean albacore populations inferred from mitochondrial and nuclear DNA markers

Genetic population structure of Atlantic and Mediterranean albacore Thunnus alalunga was investigated using nucleotide sequence variations of the glucose‐6‐phosphate dehydrogenase gene intron (G6PD) and the mitochondrial DNA (mtDNA) D‐loop region (Dloop). Restriction analysis using Ase I digestion detected two major restriction types (A and B) at the Dloop locus with strong frequency differences between Atlantic and Mediterranean samples. Thirty‐six individuals of 100 Mediterranean albacore were of the B type whereas no B type individuals were found in the Atlantic samples (n = 102). Phylogenetic analysis using nucleotide sequence data of the Dloop locus indicated that the B type lineage recently arose from the ancestral A lineage in the Mediterranean Sea and has not dispersed into the Atlantic Ocean. The frequencies of two alleles (L and S) at the G6PD locus were significantly different between the samples from the Atlantic (L = 0·495) and the Mediterranean (L = 0·725), but no significant heterogeneity was observed between mtDNA‐A and ‐B types of the Mediterranean sample. These molecular data indicate that gene flow between the Atlantic and Mediterranean albacore populations have been considerably restricted and strongly suggest these populations should continue to be treated as two distinct management units.

Mapping of X chromosome inversion breakpoints [inv(X)(q11q28)] associated with FG syndrome: A second FG locus [FGS2

FG syndrome is an X-linked condition comprising mental retardation, congenital hypotonia, macrocephaly, distinctive facial changes, and constipation or anal malformations. In a linkage analysis, we mapped a major FG syndrome locus [FGS1] to Xq13, between loci DXS135 and DXS1066. The same data, however, clearly demonstrated genetic heterogeneity. Recently, we studied a French family in which an inversion [inv(X)(q12q28)] segregates with clinical symptoms of FG syndrome. This suggests that one of the breakpoints corresponds to a second FG syndrome locus [FGS2]. We report the results of fluorescence in situ hybridization analysis performed in this family using YACs and cosmids encompassing the Xq11q12 and Xq28 regions. Two YACs, one positive for the DXS1 locus at Xq11.2 and one positive for the color vision pigment genes and G6PD loci at Xq28, were found to cross the breakpoints, respectively. We postulate that a gene might be disrupted by one of the breakpoints.

Nutrients that influence erythrocyte redox status and adaptation at the G6PD locus

Nutrients that influence erythrocyte redox status and adaptation at the G6PD locus

Long-range sequence analysis in Xq28: thirteen known and six candidate genes in 219.4 kb of high GC DNA between the RCP/GCP and G6PD loci.

DNA comprising 219 447 bp was sequenced in nine cosmids and verified at > 99.9% precision. Of the standard repetitive elements, 187 Alus make up 20.6% of the sequence, but there were only 27 MERs (2.9%) and 17 L1 fragments (1.6%). This may be characteristic of such high GC (57%) regions. The sequence also includes an 11.3 kb tract duplicated with 99.2% identity at a distance of 38 kb. The region is 80-90% transcribed and 12.5% translated. Thirteen known genes and their exon-intron borders are all accurately predicted at least in part by GRAIL programs, as are six additional genes. From centromere to telomere, the orientation of transcription varies among the first eight genes, then runs centromeric to telomeric for the next five, and is in the opposite sense for the last six. Eighteen of the 19 genes are associated with CpG islands. Two islands are exact copies in the 11.3 kb repeat units, and could thus give rise to double dosage levels of an X-linked gene. Another island is associated with two genes transcribed in opposite directions. From the sequence data, three genes and their exon structure are inferred. One of them, previously associated with HEX2, is shown to be a different gene unrelated to hexokinases; a second gene, previously known by an EST, is plexin, from its 65.5% identity with the Xenopus analog; and a third is a subunit of a vacuolar H-ATPase, and is named VATPS1.

Cloning of a gene encoding a DNase I-like endonuclease in the human Xq28 region

To contribute to the isolation of genes within the q24-qter region of the human X chromosome, we screened three cDNA libraries (human fetal brain, liver and skeletal muscle) with a cosmid clone containing a CpG island previously mapped in the q28 region. A full-length 2.1-kb cDNA clone was isolated ( XIB); DNA databank searches revealed identity with an EST fragment ( XAP-1), residing between the RCP/GCP and G6PD loci. The XIB coding region (909 bp) showed 44% amino acid (aa) identity to pig DNase I. Several conserved residues have been observed between these two genes including aa in the active site. XIB expressed a single transcript in adult heart and skeletal muscle, whereas, in some fetal tissues, two different-sized transcripts were seen. Zoo blot analysis showed a remarkable cross-species conservation. Expression and sequence of this novel gene are reported.

Independent origin of single and double mutations in the human glucose 6-phosphate dehydrogenase gene.

The vast majority of both polymorphic and sporadic G6PD variants are due to single missense mutations. In the four polymorphic variants that have two point mutations, one of the mutations is always 376 A-->G (126 Asn-->Asp), which on its own gives rise to the nondeficient polymorphic variant, G6PD A. In a study of G6PD deficient patients who presented with clinical favism in Spain, we have found a new polymorphic variant that we have called G6PD Malaga, whose only abnormality is a 542 A-->T (181 Asp-->Val) mutation. This is the same mutation as previously found in association with the mutation of G6PD A in the double mutant, G6PD Santamaria. G6PD Malaga is associated with enzyme deficiency (class III), and the enzymic properties of G6PD Malaga and G6PD Santamaria are quite similar, indicating that in this case the effects of the two mutations are additive rather than synergistic. G6PD Santamaria might have been produced by recombination between G6PD A and G6PD Malaga; however haplotype analysis, including the use of a new silent polymorphism, suggests that the same 542 A-->T mutation has taken place independently in a G6PD B gene to give G6PD Malaga and in a G6PD A gene to give G6PD Santamaria. These findings help to outline the relationship and evolution of mutations in the human G6PD locus.

Electrophoretic Study of Two Closely Related Species of North American Trichogramma: T. pretiosum and T. deion (Hymenoptera: Trichogrammatidae)

Allozymic variation was examined at 13 loci in T. pretiosum Riley and T. deion Pinto & Oatman, two cryptic species of North American Trichogramma . The 23 cultures of T. pretiosum and the 21 cultures of T. deion studied represent a significant portion of the known geographic range of both species. As with morphology, the electrophoretic data suggest close relationship. The only apparent fixed difference occurs at the G6pd locus. Syntopic collections from Riverside, CA, also differ at the Acp-II and Pgm loci, indicating that gene exchange is not occurring. Although considerable intraspecific variation occurs in these species, it is not obviously correlated with geographic distribution or mode of reproduction. Collections of T. pretiosum from Australia are similar to those from North America, suggesting that the species only recently was introduced to Australia.

Associations between Environmental Heterogeneity, Heterozygosity, and Growth Rates of Populus tremuloides in a Cordilleran Landscape

The relationship between mean annual incremental growth, habitat heterogeneity and heterozygosity at 14 polymorphic loci was studied in a natural population of trembling aspen (Populus tremuloides) in the premontane-montane region of Waterton Lakes National Park, Alberta, Canada. Multiple correlation of mean annual incremental growth in relation to elevation, aspect, exposure, slope, slope position, and age of the oldest standing ramet explained 56% of the variability in growth. A positive correlation was found between an individual tree's growth rate and its degree of heterozygosity after controlling for environmental influence. There was also a positive correlation between growth and the degree of heterozygosity when clones were grouped into heterozygosity classes. Mean annual incremental growth was most strongly associated with heterozygosity at the G6PD, ADH, ALD, and G2DH loci; growth rates were higher in heterozygotes than homozygotes. The differences in heterozygosity might be attributed to the enzyme loci involved in regulatory functions. The implications of these results for forest structure and succession are discussed.

Actin-Binding Protein (ABP-280) Filamin Gene (FLN) Maps Telomeric to the Color Vision Locus (R/GCP) and Centromeric to G6PD in Xq28

Actin-binding protein-280 (ABP-280) is a dimeric actin filament crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. We have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus.

Severe Hemophilia A in a Female by Cryptic Translocation: Order and Orientation of Factor VIII within Xq28

We report studies of a female with severe hemophilia A resulting from a complex de novo translocation of chromosomes X and 17 (46,X,t(X;17)). Somatic cell hybrids containing the normal X, the der(X), or the der(17) were analyzed for coagulation factor VIII (F8C) sequences using Southern blots and polymerase chain reaction. The normal X, always late replicating, contains a normal F8C gene, whereas the der(X) has no F8C sequences. The der(17) chromosome containing Xq24-Xq28 carries a functional G6PD locus and a deleted F8C allele that lacks exons 1-15. Also, it lacks the DXYS64-X locus, situated between the F8C locus and the Xq telomere. These results indicate that a cryptic breakpoint within Xq28 deleted the 5′ end of F8C, but left the more proximal G6PD locus intact on the der(17) chromosome. As the deleted segment includes the 5′ half of F8C as well as the subtelomeric DXYS64 locus, F8C must be oriented on the chromosome with its 5′ region closest to the telomere. Therefore, the order of these loci is Xcen-G6PD-3′F8C-5′F8C-DXYS64-Xqtel. The analysis of somatic cell hybrids has elucidated the true nature of the F8C mutation in the proband, revealing a more complex rearrangement (three chromosomes involved) than that expected from cytogenetic analysis, chromosome painting, and Southern blots. A 900-kb segment within Xq28 has been translocated to another autosome. Hemophilia A in this heterozygous female is due to the decapitation of the F8C gene on the der(17) and inactivation of the intact allele on the normal X.

Co-evolution of glucose-6-phosphate dehydrogenase deficiency and quinine taste sensitivity.

We hypothesized that 'quinine' taste sensitivity functions to regulate the intake of bitter-tasting, naturally occurring antimalarial substances of plant origin, and that this genetic trait has co-evolved with the G6PD locus. This hypothesis was tested by evaluating taste sensitivity to quinine sulphate and sodium chloride among 17 G6PD-deficient and 25 G6PD-normal African American subjects 14-40 years of age. There was no significant difference in mean 'quinine' taste sensitivity between the two groups, although there was a trend towards greater 'quinine' taste acuity among the G6PD-deficient subjects. A larger study sample would provide a fairer test of the hypothesis.

G6PD deficiency as protection againstfalciparum malaria: An epidemiologic critique of population and experimental studies

Several recent reviews in the medical literature maintain that only heterozygous G6PD deficient females are relatively protected against falciparum malaria. However, a number of population studies provide compelling evidence that both the hemizygous G6PD deficient male and homozygous G6PD deficient female are also relatively protected against falciparum parasitization. An epidemiologic critique of a sample of these field investigations points out the methodological problems that underlie some of the negative findings. In vitro studies also provide compelling evidence that erythrocytes from all G6PD deficient genotypes are relatively protected against falciparum infestation, and that this protection is enhanced by oxidant substances derived from a number of food crops such as fava beans. It is suggested that “quinine” taste sensitivity reflects taste sensitivity to bitter-tasting, naturally occurring antimalarial substances of plant origin, and that the G6PD polymorphism and the genetic loci coding for “quinine” taste sensitivity have co-evolved in human populations. It appears that adaptation at the G6PD locus in human populations reflects an intricate web of interactions between a large number of different G6PD deficient alleles which have reached polymorphic frequencies and a variety of food crops from which oxidant substances may be derived. © 1993 Wiley-Liss, Inc.

The accuracy of DNA sequences: Estimating sequence quality

In this paper we describe a method for the statistical reconstruction of a large DNA sequence from a set of sequenced fragments. We assume that the fragments have been assembled and address the problem of determining the degree to which the reconstructed sequence is free from errors, i.e., its accuracy. A consensus distribution is derived from the assembled fragment configuration based upon the rates of sequencing errors in the individual fragments. The consensus distribution can be used to find a minimally redundant consensus sequence that meets a prespecified confidence level, either base by base or across any region of the sequence. A likelihood-based procedure for the estimation of the sequencing error rates, which utilizes an iterative EM algorithm, is described. Prior knowledge of the error rates is easily incorporated into the estimation procedure. The methods are applied to a set of assembled sequence fragments from the human G6PD locus. We close the paper with a brief discussion of the relevance and practical implications of this work.

Completion of the physical map of Xq28: the location of the gene for L1CAM on the human X Chromosome

The gene for the neural cell adhesion molecule L1 (L1CAM) has been shown to be located close to the color vision pigment genes in mouse and man. This location has been confirmed by a number of different mapping strategies in both species. With pulsed field gel electrophoresis it has been proposed that L1CAM lies between the RCP, GCP, and GDX, G6PD loci. We report here a reinterpretation of the location of this gene, based on the physical linkage of L1CAM to the more proximal locus DXS15. This places L1CAM between this marker and the color vision genes (RCP, GCP), a region very dense in CpG islands, expected to contain a large fraction of the disease genes assigned to the Xq28 region. In combination with the physical mapping data on Xq28 described previously, this closes the last remaining gap in the map of the Xq27-Xq28 region. This removes the last contradiction between the maps of this region in the genomes of man and mouse, and confirms the close similarity of order and distances of markers between these organisms.