A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

et al. (2014) A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya. PLoS ONE 9(5): e96486. doi:10.1371/journal.pone.0096486 A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya Jason P. Wendler 0 John Okombo 0 Roberto Amato 0 Olivo Miotto 0 Steven M. Kiara 0 Leah Mwai 0 Lewa Pole 0 John O'Brien 0 Magnus Manske 0 Dan Alcock 0 Eleanor Drury 0 Mandy Sanders 0 Samuel O. Oyola 0 Cinzia Malangone 0 Dushyanth Jyothi 0 Alistair Miles 0 Kirk A. Rockett 0 Bronwyn L. MacInnis 0 Kevin Marsh 0 Philip Bejon 0 Alexis Nzila 0 Dominic P. Kwiatkowski 0 Stuart Alexander Ralph, University of Melbourne, Australia 0 1 Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford , Oxford , United Kingdom , 2 Kenya Medical Research Institute (KEMRI)/ Wellcome Trust Collaborative Research Program, Kilifi, Kenya, 3 Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University , Bangkok , Thailand , 4 Wellcome Trust Sanger Institute , Hinxton, Cambridge , United Kingdom , 5 Wellcome Trust Centre for Human Genetics, University of Oxford , Oxford , United Kingdom , 6 Department of Biology, King Fahd University of Petroleum and Minerals , Dhahran , Saudi Arabia Background: 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. Methods and Principal Findings: 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. - Funding: This research was supported by the Medical Research Council (G0600718) and The Wellcome Trust (090770/Z/09/Z). The Wellcome Trust also provide core awards to Wellcome Trust Centre for Human Genetics (090532/Z/09/Z) and the Wellcome Trust Sanger Institute (098051). http://www.wellcome.ac.uk/index. htm http://www.mrc.ac.uk/index.htm The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Philip Bejon and Alexis Nzila are PLOS ONE editorial board members. This does not alter the authors adherence to PLOS ONE Editorial policies and criteria. . These authors contributed equally to this work. " These authors are joint last authors on this work. Plasmodium falciparum malaria is responsible for nearly 600,000 African deaths every year, and in Kenya consumes a fifth of hospitalization resources [1,2]. Prompt treatment with antimalarials can prevent mortality, but this efficacy is threatened by the parasites ability to acquire drug resistance. This highlights the appeal of high-resolution genetic markers and data-sharing for early-warning surveillance [3]. Additionally, the elucidation of genetic loci underlying resistance is important for designing new formulations, and can reveal opposing selective pressures amongst drugs [4]. Drug resistance loci in P. falciparum parasites have been discovered using genetic crosses for QTL analysis [5,6]. A number of recent studies targeted on particular parasite genes in coastal Kenya have described drug activity associations with familiar SNPs in pfmdr1, pfcrt, and pfdhfr, as well as structural associations with quinine (QN) tolerance in pfnhe [7,8,9]. Population-genetic approaches, such as sequence-based GWAS, provide the advantage of testing for phenotypic associations with novel SNPs while broadly surveying known polymorphisms [10]. This work examines the association between SNPs ascertained from whole-genome sequencing of 35 Kenyan field isolates with the activities of 22 antimalarial drugs (Figure S1, Table S1). The cooperative efforts of the partnerships in the Malaria Genomic Epidemiology Network (MalariaGEN) have created a panel of highly credible SNPs ascertained in the context of 1685 parasites, contributed from 17 countries, and we utilize this community resource here [11]. Materials and Methods Ethics statement Parasites were isolated from the peripheral blood of participants in two clinical trials on Artekin versus Coartem, conducted in Kilifi between 2005 and 2007. All studies obtained clearance from the Kenya Medical Research Institute (KEMRI) Ethical Review Committee under the protocol numbers SSC 945 and SSC 946. Sample collection and processing Infected blood pellets were cryopreserved using glycerolyte and later adapted to culture as described elsewhere [12]. Pellets were frozen for three months on average before culture adaptation and chemosensitivity testing, and were in continuous culture for approximately two months for these assays before DNA extraction and sequencing (Figure S2). DNA was extracted from adapted field isolates using the QIAamp DNA Blood Mini Kit (Qiagen, UK). Of the thirty-five isolates used in the final analysis, thirteen were taken from patients admitted to Kilifi District Hospital with severe malaria, and twenty-two from participants in a study comparing Artekin to Coartem [13]. Of these latter twenty-two, twelve were collected at recruitment, and ten were collected 1984 days later (mean = 48.7days), representing reinfections or recrudescences. Two of the ten follow-up samples are from patients also represented at recruitment in this dataset. We classified both of these cases as reinfections because, based on the number of SNP identities, the recruitment and follow-up parasites were no more similar to one another than to those from other patients. Chemosensitivity testing Detail (...truncated)