Wendler Jason P, Okombo John, Amato Roberto, Miotto Olivo, Kiara Steven M, Mwai Leah, Pole Lewa, O'Brien John, Manske Magnus, Alcock Dan, Drury Eleanor, Sanders Mandy, Oyola Samuel O, Malangone Cinzia, Jyothi Dushyanth, Miles Alistair, Rockett Kirk A, MacInnis Bronwyn L, Marsh Kevin, Bejon Philip, Nzila Alexis, Kwiatkowski Dominic P
Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom; Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research Program, Kilifi, Kenya.
PLoS One. 2014 May 8;9(5):e96486. doi: 10.1371/journal.pone.0096486. eCollection 2014.
Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.
Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.
CONCLUSIONS/SIGNIFICANCE: Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.
耐药性仍然是疟疾控制的主要关注点。为了确定耐药寄生虫的遗传标记，我们测试了来自35个肯尼亚恶性疟原虫寄生虫的基于序列的基因型与22种抗疟药物活性之间的全基因组关联（GWA）。
从患有急性发热性疟疾的儿童中分离出的寄生虫适应培养，并通过在抗疟药物存在下的体外生长来确定敏感性。使用全基因组测序技术对寄生虫进行基因分型。确定了6250个单核苷酸多态性（SNP）与对个体抗疟药物耐药性之间的关联，并对多重假设检验进行了错误发现率调整。我们在pfcrt区域发现了与氯喹（CQ）活性相关的预期关联，以及与阿莫地喹、喹唑啉和奎宁活性相关的其他新位点。CQ和伯氨喹（PQ）的信号在pfcrt及其周围重叠，有趣的是，这两种药物的表型呈负相关。我们对dhfr、dhps、mdr1、nhe和crt中的变异进行了编目，包括新的SNP，并证实了肯尼亚沿海地区存在dhfr - 164L四重突变体。在该样本集中，与磺胺多辛 - 乙胺嘧啶耐药性相关的突变处于或接近固定状态。
结论/意义：基于序列的GWA研究是进行表型关联测试的强大工具。通过对肯尼亚沿海地区的恶性疟原虫采用这种方法，我们确定了与抗疟药物表型耐药性相关的已知和先前未报道的基因，并在高分辨率单倍型可视化中观察到CQ和PQ之间可能存在反向选择关系的特征。
