Questions over high frequency of mutant PfATP6 haplotypes in traveller isolates

Progress in malaria control over the past decade has been striking, with malaria mortality rates falling by approximately one quarter globally and more than a third in the World Health Organization African Region. In the accompanying paper, Cohen et al. demonstrate the potential fragility of these gains, comprehensively describing malaria resurgences that have occurred over the past 80 or so years. They found that the vast majority of resurgences were due, at least in part, to the weakening of malaria control programmes; resource constraints were the most commonly identified factor. Their findings are timely and compelling, demonstrating that global efforts will be wasted if the required resources are not secured to achieve and maintain universal access to life-saving malaria prevention and control tools. The greatest threats to current malaria control efforts are not biological, but financial. The increases in funding for malaria over the past decade, while impressive, still fall far short of the nearly $6 billion dollars required annually. Domestic spending by endemic country governments on malaria specifically, and health more generally, could go a long way towards filling the projected funding gap. However, external funding is also essential, and the global community needs to work together to ensure full funding of the Global Fund to Fight AIDS, Tuberculosis, and Malaria, which has been the single largest source of malaria funding over the past decade. This year, on April 25th, World Malaria Day will be celebrated with the theme Sustain Gains, Save Lives: Invest in Malaria. The review by Cohen et al. suggests one possible future if such investment is not made. However, with sufficient support, malaria resurgences can be relegated to history.     

Three-dimensional structure of Plasmodium falciparum Ca2+ -ATPase(PfATP6) and docking of artemisinin derivatives to PfATP6

Construction of the 3D structure of PfATP6 by homology modeling and docking simulation of artemisinin derivatives to this protein model are reported. Docking and consequent LUDI scores show good relation with in vitro antimalarial activities. The main binding source of artemisinins to the PfATP6 is hydrophobic interaction and biologically important peroxide bonds were exposed to outside of the binding pocket. This study suggests binding of artemisinin to PfATP6 precedes activation of peroxide bond by Fe(2+) species.     

PAX6 haplotypes are associated with high myopia in Han chinese

Background

The paired box 6 (PAX6) gene is considered as a master gene for eye development. Linkage of myopia to the PAX6 region on chromosome 11p13 was shown in several studies, but the results for association between myopia and PAX6 were inconsistent so far.

Methodology/Principal Findings

We genotyped 16 single nucleotide polymorphisms (SNPs) in the PAX6 gene and its regulatory regions in an initial study for 300 high myopia cases and 300 controls (Group 1), and successfully replicated the positive results with another independent group of 299 high myopia cases and 299 controls (Group 2). Five SNPs were genotyped in the replication study. The spherical equivalent of subjects with high myopia was ≤−8.0 dioptres. The PLINK package was used for genetic data analysis. No association was found between each of the SNPs and high myopia. However, exhaustive sliding-window haplotype analysis highlighted an important role for rs12421026 because haplotypes containing this SNP were found to be associated with high myopia. The most significant results were given by the 4-SNP haplotype window consisting of rs2071754, rs3026393, rs1506 and rs12421026 (P = 3.54×10⁻¹⁰, 4.06×10⁻¹¹ and 1.56×10⁻¹⁸ for Group 1, Group 2 and Combined Group, respectively) and the 3-SNP haplotype window composed of rs3026393, rs1506 and rs12421026 (P = 5.48×10⁻¹⁰, 7.93×10⁻¹² and 6.28×10⁻²³ for the three respective groups). The results remained significant after correction for multiple comparisons by permutations. The associated haplotyes found in a previous study were also successfully replicated in this study.

Conclusions/Significance

PAX6 haplotypes are associated with susceptibility to the development of high myopia in Chinese. The PAX6 locus plays a role in high myopia.     

Status of potential PfATP6 molecular markers for artemisinin resistance in Suriname

ABSTRACT: BACKGROUND: Polymorphisms within the PfATP6 gene have been indicated as potential molecular markers for artemisinin efficacy. Since 2004, the use of artemisinin combination therapy (ACT) was introduced as first-line treatment of the uncomplicated malaria cases in Suriname. The aim of this research was to determine changes in Suriname in the status of the polymorphic markers in the PfATP6 gene before and after the adoption of the ACT-regimen, particularly of the S769N mutation, which was reported to be associated with in vitro Artemether resistance in the neighboring country French Guiana. METHODS: The PfATP6 gene from Plasmodium falciparum parasites in Suriname was investigated in 28 samples using PCR amplification and restriction enzyme analysis, to assess and determine the prevalence of potentially interesting single nucleotide polymorphisms. The polymorphisms [L263E; A623E; S769N], which may be associated with the artemisinin resistant phenotype were characterized in parasites from three endemic regions before and after the adoption of the ACT-regimen. In addition, the status of these molecular markers was compared in paired P. falciparum isolates from patients with recurring malaria after controlled ACT. RESULTS: All the investigated samples exhibit the wild-type genotype at all three positions; L263, A623, S769. CONCLUSION: All investigated isolates before and after the adoption of the ACT-regimen and independent of endemic region harbored the wild-type genotype for the three investigated polymorphisms. The study revealed that decreased artemisinin susceptibility could occur independent from PfATP6 mutations, challenging the assumption that artemisinin resistance is associated with these mutations in the PfATP6 gene.     

Hemoglobin Koya Dora: high frequency of a chain termination mutant.

Approximately 10% of the members of the Koya Dora tribe from Andhra Pradesh (India) carry an alpha chain hemoglobin variant, Hb Koya Dora (Hb KD), usually in amounts of 0.5%-2% of total hemoglobin. In four presumed homozygotes for Hb KD, up to 10% of the abnormal hemoglobin was present. The alpha chain of Hb KD was found to be elongated by at least 16 residues, possibly as a result of a mutation of the normal alpha chain termination codon UAA TO UCA, coding for serine. A pedigree in which two individuals possess Hb KD as well as the alpha chain variant Hb Rampa and normal Hb A proves the existence of two alpha chain loci in this population. Hb DK resembles the previously described Hb Constant Spring [6, 7] in many aspects, probably also in its alpha thalassemia-like expression.     

EMS-induced mutant frequency and spectrum in bone marrow of D6-2 transgenic mice

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A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit- mutant of Saccharomyces cerevisiae

In cultures of the mit- mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit- cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a 32P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the ori1 sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

Characterization of a timing mutant of Dictyostelium discoideum which exhibits "high frequency switching"

The preaggregative period of Dictyostelium discoideum is composed of two sequential rate-limiting components. The timing mutant FM-1 exhibits a decrease in the length of the preaggregative period and the interval between the maxifinger and early culminate II stage. In contrast, it is normal in all aspects of growth, in the sequence of morphogenetic stages, in spore formation, in the capacity to rapidly recapitulate morphogenesis, and in the erasure event and subsequent program of dedifferentiation. By the reciprocal shift experiment, it is demonstrated that FM-1 is completely missing the first of the two rate-limiting components comprising the preaggregative period. The FM-1 mutation is heritable and behaves as a single mutation mapping to linkage group II. However, the FM-1 variant switches at relatively high frequency to several other timing phenotypes with longer preaggregative periods which in turn switch at high frequency. The FM-1 phenotype is considered in terms of timing regulation, and the process of high frequency switching between timing phenotypes is compared to other newly discovered switching systems.     

Combined receptor-based and ligand-based approach to delineate the mode of binding of guaianolide-endoperoxides to PfATP6

Plasmodium falciparum calcium-ATPase (PfATP6) has been reported to be a target of artemisinin and related endoperoxides. In this study, a series of previously reported guaianolide-endoperoxides (thaperoxides) were docked into a homology model of PfATP6 and also used to develop a pharmacophore model. This combined approach led to useful insights into the binding determinants of thaperoxides to the malarial enzyme. In addition, in silico mutagenesis and molecular dynamics suggested the importance of Phe264 and the electrostatic interactions between Lys260 in helix H3 and Lys1036 and Asp1038 in L6/7 loop for the binding of thaperoxides. These results could help in the design of more potent inhibitors of PfATP6.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.     

Intrachromosomal gene conversion frequency in the H2 differs between haplotypes

 We examined two intrachromosomal gene conversion events with a polymerase chain reaction assay at the DNA level between the two major histocompatibility complex class II genes Eb and Ab in mice sperm before selection has occurred. The frequency of the intrachromosomal gene conversion event between Ebd and Abd was found to be at least one order of magnitude higher than between Ebk and Abk in the same mice. Parental imprinting of the genes appears not to have an effect on gene conversion, as both (d×k)F₁ and (k×d)F₁ mice have indistinguishable frequencies in both haplotypes. The number of DNA copies of the donor and acceptor genes present in the cell at the time of mutation does not seem to influence the frequency of the intrachromosomal gene conversion in the k haplotype, whereas the frequency in the d haplotype is increased when double the number of donor and acceptor genes is present. The DNA fragment transferred between Ebd and Abd is invariably short, and need not comprise more than six nucleotides. The fragment transferred within the k haplotype varies in length, and can attain at least 100 nucleotides. The difference between the haplotypes both in length and frequency might be attributed to a six-nucleotide deletion in the Abk gene, which might make base-pairing between the genes less efficient and less precise. Received: 24 October 1997 / Revised: 5 January 1998     

Interactions of curcumin with the PfATP6 model and the implications for its antimalarial mechanism

Despite curcumin has been proved to possess antimalarial effects, the underlying mechanism remains to be elucidated. In this letter, the active site binding modes of curcumin in PfATP6, an important antimalarial target, were investigated using computational docking. It was revealed that curcumin interacts with PfATP6 mainly through hydrophobic interactions and hydrogen bonds. Moreover, the theoretically predicted binding affinity implies that curcumin can efficiently inhibit PfATP6, which gains some deeper insights into the antimalarial mechanism of curcumin.     

In vivo mutant frequency rises among breast cancer patients after exposure to high doses of gamma-radiation

Human in vivo mutant frequencies can be measured by cloning freshly isolated lymphocytes in selective media containing 6-thioguanine (TG). This method was applied to monitoring environmental mutagenesis, by studying lymphocytes separated from peripheral blood of 12 cancer patients undergoing radiotherapy. Before therapy, cancer patients had an average 8.6 X 10(-6) mutants/cell, compared to 2.4 X 10(-6) mutants/cell for heart patients and 1.1 X 10(-6) mutants/cell for healthy controls. After exposure of cancer patients to 50 Gy of gamma-radiation delivered to the treated area, or an estimated 4 Gy received by each lymphocyte, patients averaged 36.8 X 10(-6) mutants/viable cell.     

DNA repair and mutant frequency in schizophrenia.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes was determined with a cloning assay. T-lymphocytes were obtained from 14 individuals diagnosed with schizophrenia and 5 controls. No significant difference in mutant frequency was observed between the 2 groups. In addition, DNA-repair capacity was measured with the unscheduled DNA synthesis technique in lymphocytes from 7 individuals diagnosed with schizophrenia and 7 controls. Repair capacity was determined following treatment with MMS, MNNG, and 20 J/m2 ultraviolet light. No significant differences in DNA repair were observed between the patient and control groups in response to any of the 3 DNA-damaging agents. These results argue against differences between normal and schizophrenic individuals with respect to in vivo mutant frequency or their capacity to repair DNA lesions induced by MMS, MNNG, or ultraviolet radiation.     

Lymphocyte Hprt mutant frequency and sperm toxicity in C57BL/6 mice treated chronically with Azathioprine

Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.     

Dielectric behavior of wild-type yeast and vacuole-deficient mutant over a frequency range of 10 kHz to 10 GHz.

Dielectric behavior of Saccharomyces cerevisiae wild-type and vacuole-deficient mutant cells has been studied over a frequency range of 10 kHz to 10 GHz. Both types of cells harvested at the early stationary growth phase showed dielectric dispersion that was phenomenologically formulated by a sum of three separate dispersion terms: beta 1-dispersion (main dispersion) and beta 2-dispersion (additional dispersion) and gamma-dispersion due to orientation of water molecules. The beta 1-dispersion centered at a few MHz, which has been extensively studied so far, is due to interfacial polarization (or the Maxwell-Wagner effect) related to the plasma membrane. The beta 2-dispersion for the vacuole-deficient mutant centered at approximately 50 MHz was explained by taking the cell wall into account, whereas, for the wild-type cells, the beta 2-dispersion around a few tens MHz involved the contributions from the vacuole and cell wall.     

Identification of glucose 6-phosphate dehydrogenase deficiency in a population with a high frequency of thalassemia

High frequencies of both thalassemia trait (5.2%) and glucose 6-phosphate dehydrogenase (G6PD) deficiency for only males (1.3%) have been observed in the Calabrian population. The G6PD activity measurement was carried out on 1239 samples of whole blood from Calabrian subjects of both sexes (age range 10-55) by a differential pH-metry technique which was quite suitable to determine the G6PD deficiency in mass screenings. The analyzed subjects showed: only the thalassemia trait; or only the G6PD deficiency; or only the total iron serum deficiency; or G6PD deficiency associated with the thalassemia trait or with the total iron serum deficiency. The G6PD heterozygous subjects have an enzymatic activity which is masked by both the thalassemia trait and the total iron serum deficiency. In a population showing high frequencies of both thalassemia trait and G6PD deficiency, the comparison of G6PD activity of heterozygous subjects also affected with the thalassemia trait is more reliable if referred to the enzymatic activity of the carriers of the latter inherited anomaly rather than to G6PD activity of normal subjects.     

High-throughput analysis of informative CYP2D6 compound haplotypes

We describe a high-throughput protocol for detecting key polymorphisms in the drug-metabolizing enzyme gene CYP2D6 and a number of linked microsatellites that is both fast and relatively inexpensive to perform. This approach employs GeneScan technology to enable a researcher to determine rapidly the status of seven simple nucleotide polymorphisms in CYP2D6 and also to assay repeat number variation at five closely linked dinucleotide microsatellite loci. The method requires only three PCRs and two GeneScan runs per sample. We anticipate that this will be of value to researchers in three different ways: (1) rapid discrimination of common CYP2D6 alleles, (2) high-resolution haplotyping for association studies involving chromosome 22q13.1 using microsatellite variation, and (3) generation of compound haplotypes for investigating the evolution of CYP2D6 variation. We also report compound haplotype frequencies for an Ashkenazi Jewish and a British sample.