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Open Access Research article

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

Katarzyna Kinga Modrzynska18, Alison Creasey1, Laurence Loewe2, Timothee Cezard3, Sofia Trindade Borges49, Axel Martinelli5, Louise Rodrigues105, Pedro Cravo115, Mark Blaxter36, Richard Carter1 and Paul Hunt17*

Author Affiliations

1 Institute for Immunology and Infection Research, University of Edinburgh, Edinburgh, UK

2 Laboratory of Genetics and Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, USA

3 The GenePool, University of Edinburgh, Edinburgh, UK

4 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Malaria, Lisbon, Portugal

5 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular, Lisbon, Portugal

6 Institute for Evolutionary Biology, University of Edinburgh, Edinburgh, UK

7 Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK

8 Current address: The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK

9 Current address: Research Unit and Cardiology department, Funchal Hospital Center, Funchal. Madeira, Portugal

10 Current address: Microbiology, Molecular Genetics and Immunology, Kansas University Medical Center, Kansas City, USA

11 Current address: IPTSP, Universidade Federal de Goiás, Goiânia, Brasil

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BMC Genomics 2012, 13:106  doi:10.1186/1471-2164-13-106

Published: 21 March 2012

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.