Mapping Drug Resistance Genes in Plasmodium falciparum

描述(申请人提供)：青蒿素联合疗法(ACTS)是治疗恶性疟原虫的主要方法。然而，在一些东南亚国家，在使用ACTS治疗后，寄生虫从血液中清除的速度很慢，这引发了人们对即将出现抗药性的担忧。这一更新应用的中心目标是识别导致青蒿素(ART)治疗后慢清除率(CR)的寄生虫基因，了解这一特征的进化，并通过转基因探索潜在的机制。为了确定慢缓解率的标志，我们将与泰国-缅甸边境的疟疾寄生虫合作，因为该地区的患者在接受ART治疗后表现出广泛的缓解率，我们已经证明，缓解率的大部分(58%)变异可以由寄生虫遗传因素解释。我们将使用自2007年以来收集的650个指刺寄生虫DNA样本进行分析。这些样本在遗传上是独一无二的(来自96个SNP的基因分型)，含有单一的疟疾基因型，并具有强大的(每小时6个)寄生虫清除率指标。我们将使用专门为东南亚寄生虫设计的NimbleGen微阵列，对这些样本进行16,875个多态单核苷酸多态(SNPs)的基因分型，从泰国参考寄生虫种群(已获得101个全基因组序列)中推断出额外的SNPs，并使用全基因组关联研究(GWAS)确定CR背后的基因。为了证实这些基因座的参与，我们将对来自6个东南亚国家的12个独立寄生虫种群中确定的候选基因进行基因分型，从泰国和柬埔寨进行10年的纵向采样。这些数据将使我们能够验证来自GWAs的关联，确定致病等位基因的分布，跟踪候选基因座的等位基因频率随时间的变化，并确定该性状的独立起源的数量。目前还没有良好的表型分析缓慢的CR性状用于实验室。我们将评估两种有希望的方法的实用性，一种是定量复发试验，另一种是基于流式细胞术的生长试验，它们有效地区分实验室选择的抗逆转录病毒寄生虫及其敏感的祖细胞。这将使用从泰国和缅甸边境缓慢和快速清除寄生虫的面板来完成，这些寄生虫可以获得冷冻保存的库存。最后，为了确定因果关系并研究慢CR的潜在机制，我们将操纵候选基因的表达，并检查这如何改变与慢CR表型相关的体外替代措施。有效的伙伴药物对于维持使用ACTS的有效治疗至关重要。我们还将使用转染法来检查伙伴药物耐药的候选标记，这些标记是在前一次赠款期间进行的一项GWAS研究中确定的。

DESCRIPTION (provided by applicant): Artemisinin combination therapies (ACTs) are the mainstay of treatment for Plasmodium falciparum. However, slow clearance of parasites from the blood following treatment with ACTs in some SE Asian countries has raised fears of impending resistance. The central goals of this renewal application are to identify parasite genes that underlie slow clearance rate (CR) following treatment with artemisinin (ART), to understand the evolution of this trait, and to probe the underlying mechanisms using transfection. To identify markers for slow CR, we will work with malaria parasites from the Thai-Burma border, because patients in this region show a wide range of CR following ART treatment, and we have shown that the most (58%) of the variation in CR can be explained by parasite genetic factors. We will use 650 finger-prick parasite DNA samples collected since 2007 for this analysis. These samples are genetically unique (from genotyping 96 SNPs), contain single malaria genotypes and have robust (6 hourly) measures of parasite clearance rate. We will genotype these samples at 16,875 polymorphic single nucleotide polymorphisms (SNPs) using a Nimblegen microarray specifically designed for SE Asian parasites, impute additional SNPs from a Thai reference parasite population for which 101 whole genome sequences are available, and identify the genes that underlie CR using a genome wide association study (GWAS). To confirm involvement of these loci, we will genotype candidate genes identified in 12 independent parasite populations from six SE Asian countries, with 10 year longitudinal sampling from Thailand and Cambodia. These data will allow verification of associations from our GWAS, determine the distribution of causative alleles, track changes in allele frequency of candidate loci over time, and identify numbers of independent origins of this trait. There are currently no good phenotypic assays of the slow CR trait for use in the laboratory. We will evaluate the utility of two promising measures, a quantitative recrudescence assay and a flow cytometry-based growth assay, which effectively differentiate between ART resistant parasites selected in the laboratory and their sensitive progenitors. This will be done using panels of slow and fast clearing parasites from the Thai-Burma border, for which cryopreserved stocks are available. Finally, to determine causality and investigate the underlying mechanisms of slow CR, we will manipulate expression of candidate genes and examine how this alters surrogate in vitro measures associated with the slow CR phenotype. Effective partner drugs are critical for maintaining effective treatment using ACTs. We will also use transfection to examine candidate markers for resistance to partner drugs identified from a GWAS study conducted during the previous grant period.