Mapping Drug Resistance Genes in Plasmodium falciparum

描述（由申请方提供）：青蒿素联合疗法（ACT）是治疗恶性疟原虫的主要方法。然而，在一些东南亚国家，用青蒿素综合疗法治疗后，寄生虫从血液中清除缓慢，这引起了人们对即将出现耐药性的担忧。这项更新申请的中心目标是确定青蒿素（ART）治疗后清除率（CR）缓慢的寄生虫基因，以了解这种特性的演变，并使用转染探索潜在的机制。为了确定缓慢CR的标志物，我们将与来自泰缅边境的疟疾寄生虫合作，因为该地区的患者在ART治疗后显示出广泛的CR，并且我们已经表明，CR中的大多数（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份手指刺血寄生虫DNA样本进行分析。这些样本在遗传上是独特的（来自基因分型96个SNP），含有单一疟疾基因型，并具有稳健的（6小时）寄生虫清除率测量。我们将使用专为东南亚寄生虫设计的Nimblegen微阵列对这些样本进行16，875个多态性单核苷酸多态性（SNP）的基因分型，从泰国参考寄生虫人群（可获得101个全基因组序列）中估算额外的SNP，并使用全基因组关联研究（GWAS）鉴定CR的基础基因。为了确认这些位点的参与，我们将在12个独立的寄生虫种群从东南亚6个国家，10年的纵向抽样从泰国和柬埔寨的基因型候选基因。这些数据将允许验证来自我们的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测定的慢CR性状在实验室中使用。我们将评估两个有前途的措施，定量复发测定和流式细胞术为基础的生长测定，有效区分在实验室中选择的抗ART寄生虫和它们的敏感祖细胞的效用。这将使用来自泰缅边境的缓慢和快速清除寄生虫的面板来完成，其中冷冻保存的股票可用。最后，为了确定因果关系并研究缓慢CR的潜在机制，我们将操纵候选基因的表达，并研究这如何改变与缓慢CR表型相关的体外替代指标。有效的伙伴药物对于维持青蒿素综合疗法的有效治疗至关重要。我们还将使用转染来检查在前一个资助期进行的GWAS研究中确定的对合作药物耐药的候选标记。

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.