Validation of Pyrosequencing for accurate and high throughput estimation of allele frequencies in malaria parasites.

Institute for Immunology and Infection Research, School of Biological Sciences, The University of Edinburgh, Ashworth Laboratories, The Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK.
Molecular and Biochemical Parasitology (Impact Factor: 2.24). 05/2007; 152(2):213-9. DOI: 10.1016/j.molbiopara.2006.12.008
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    ABSTRACT: If drug-resistant malaria mutants are less fit than sensitive forms, they will wane over time when active drug pressure is removed and the overall sensitivity to the drug may be restored. However, most studies addressing this issue have been largely retrospective. Here, we undertook a predictive study, using mutant rodent malaria parasites resistant to the Artemisinin Combination Treatment (ACT) version of artesunate + mefloquine (ATN + MF) to gain insights about their ability to compete with ATN + MF-sensitive forms in untreated hosts. Previously, Plasmodium chabaudi parasites resistant to ATN + MF were selected in vivo through prolonged passaging in mice under increasing doses of the two drugs, and shown to harbour duplication of the mdr1 gene. Here, the resistant parasite, AS-ATNMF1, was mixed with its progenitor AS-ATN in different proportions and each mixture was injected into mice that were left untreated. Absolute percentage parasitaemias and the proportion of each parasite were then monitored by microscopy and proportional sequencing, respectively, every two days for a period of 14 days. AS-ATNMF1 outperformed its progenitor AS-ATN over the whole sampling period regardless of the relative starting proportion of each parasite clone. In order to assess if consecutive sub-inoculations could have been responsible for the apparent fitness gain of the resistant parasite, its growth was compared to that of AS-ATN27P, a parasite which was passaged the same number of times as AS-ATNMF1, but left untreated. Although small fluctuations in the proportion of each parasite were observed through time, the relative abundance of each on the last day of sampling (Day 14) was virtually identical to that of the starting inoculum. We conclude that there is no fitness cost associated with MDR1-associated ATN + MF resistance in vivo. These observations offer the first insights about the within-host dynamics between ACT-resistant and -sensitive parasites in absence of drug pressure.
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    ABSTRACT: Background.Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug specific cost-benefit index, indicating the possible advantage for mutated parasites.Methods.We specifically studied pfmdr1 D1246Y in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine and chloroquine, as well as relative fitness were determined in two modified isogenic P. falciparum clones only differing in the pfmdr1 1246 position. Data were used to create a new comparative graph of the relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment.Results.Pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine, but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine exposed parasites. In vivo a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data.Conclusions.This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.
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    ABSTRACT: Constitutional DICER1 mutations have been associated with pleuropulmonary blastoma, cystic nephroma, Sertoli-Leydig tumours and multinodular goitres, while somatic DICER1 mutations have been reported in additional tumour types. Here we report a novel syndrome termed GLOW, an acronym for its core phenotypic findings, which include Global developmental delay, Lung cysts, Overgrowth and Wilms tumour caused by mutations in the RNase IIIb domain of DICER1. We performed whole exome sequencing on peripheral mononuclear blood cells of an affected proband and identified a de novo missense mutation in the RNase IIIb domain of DICER1. We confirmed an additional de novo missense mutation in the same domain of an unrelated case by Sanger sequencing. These missense mutations in the RNase IIIb domain of DICER1 are suspected to affect one of four metal binding sites located within this domain. Pyrosequencing was used to determine the relative abundance of mutant alleles in various tissue types. The relative mutation abundance is highest in Wilms tumour and unaffected kidney samples when compared with blood, confirming that the mutation is mosaic. Finally, we performed bioinformatic analysis of microRNAs expressed in murine cells carrying specific Dicer1 RNase IIIb domain metal binding site-associated mutations. We have identified a subset of 3p microRNAs that are overexpressed whose target genes are over-represented in mTOR, MAPK and TGF-β signalling pathways. We propose that mutations affecting the metal binding sites of the DICER1 RNase IIIb domain alter the balance of 3p and 5p microRNAs leading to deregulation of these growth signalling pathways, causing a novel human overgrowth syndrome.
    Journal of Medical Genetics 03/2014; · 5.64 Impact Factor