title: Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.
abstract: BACKGROUND
  Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.
  
  
  RESULTS
  Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.
  
  
  CONCLUSIONS
  Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.
authors.affiliation: Institute for Immunology and Infection Research, University of Edinburgh, UK.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kinga Modrzynska
authors.forename: Alison
authors.initials: A
authors.lastname: Creasey
authors.forename: Laurence
authors.initials: L
authors.lastname: Loewe
authors.forename: Timothee
authors.initials: T
authors.lastname: Cezard
authors.forename: Sofia
authors.initials: S
authors.lastname: Trindade Borges
authors.forename: Axel
authors.initials: A
authors.lastname: Martinelli
authors.forename: Louise
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Pedro
authors.initials: P
authors.lastname: Cravo
authors.forename: Mark
authors.initials: M
authors.lastname: Blaxter
authors.forename: Richard
authors.initials: R
authors.lastname: Carter
authors.forename: Paul
authors.initials: P
authors.lastname: Hunt
mesh_terms: D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000235:genetics; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D002738:Chloroquine / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004351:Drug Resistance / Q000235:genetics*; D005260:Female; D056426:Genetic Loci / Q000235:genetics; D023281:Genomics; D006801:Humans; D008288:Malaria / Q000469:parasitology*; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D016788:Plasmodium chabaudi / Q000187:drug effects / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics; D051381:Rats; D054875:Ubiquitination / Q000235:genetics
chemical_list: D026905:Amino Acid Transport Systems; D000962:Antimalarials; D015800:Protozoan Proteins; D002738:Chloroquine