Mapping Drug Resistance Genes in Plasmodium falciparum

描述（由申请人提供）：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，在接受以青蒿素为基础的联合治疗后，血液中寄生虫的清除速度缓慢，这引起了人们对即将出现耐药性的担忧。这项更新应用的中心目标是鉴定在接受青蒿素（ART）治疗后导致清除率低（CR）的寄生虫基因，了解这一特性的进化，并利用转染探索其潜在机制。为了确定慢CR的标志物，我们将研究泰缅边境的疟疾寄生虫，因为该地区的患者在接受抗逆转录病毒治疗后出现了广泛的CR，我们已经证明，CR的大部分变异（58%）可以用寄生虫的遗传因素来解释。我们将使用自2007年以来收集的650份手指刺虫DNA样本进行分析。这些样本具有遗传独特性（来自96个snp的基因分型），包含单一疟疾基因型，并且具有可靠的（6小时）寄生虫清除率测量值。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个多态性单核苷酸多态性（snp）的基因分型，从泰国参考寄生虫种群中推导出101个全基因组序列，并使用基因组广泛关联研究（GWAS）确定导致CR的基因。为了证实这些基因座的参与，我们将对来自东南亚6个国家的12个独立寄生虫种群中鉴定的候选基因进行基因分型，并从泰国和柬埔寨进行了10年的纵向抽样。这些数据将验证GWAS的关联，确定致病等位基因的分布，跟踪候选基因座等位基因频率随时间的变化，并确定该性状的独立起源数量。目前在实验室中还没有很好的慢CR性状表型测定方法。我们将评估两种有前景的方法的效用，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，这两种方法可以有效区分实验室中选择的抗逆转录病毒耐药性寄生虫及其敏感的祖细胞。这将通过从泰缅边境缓慢和快速清除寄生虫的小组来完成，这些寄生虫有冷冻储备。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，并研究这如何改变与慢CR表型相关的体外替代指标。有效的伴用药对于维持以青蒿素为基础的联合治疗的有效治疗至关重要。我们还将使用转染技术来检测从上一个资助期进行的GWAS研究中确定的对伴侣药物耐药的候选标记物。

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.