Identification of genes influencing artemisinin and chloroquine resistance by Linkage Group Selection

About 1 million children die each year from malaria parasites, mainly in Africa. One important reason is that mutations have made the parasite resistant to drugs like chloroquine. Artemisinin is therefore a vitally important drug. It would be a major public-health disaster if resistance to artemisinin develops. We therefore need to know in advance which genes are likely to mutate.
We have already produced parasites which are resistant to artemisinin, and now wish to identify the genetic loci involved. We can do this using cutting-edge and highly efficient genetics strategies and methods. Because of the experimental and ethical limitations in using human malaria, we use a malaria species which infects mice. Other scientists will be interested in confirming our results in human malaria parasites in the laboratory and in natural infections.
This project addresses a vital question in contemporary malaria science and is likely to make a significant practical impact. It will help us to decide which drugs should be used in specific regions of the world, improve our understanding of how artemisinin drugs work, how resistance evolves and how we may reduce the rate at which resistance spreads. It will help us to design other effective drugs.

Malaria is becoming an increasing burden upon human health, largely because of the spread of drug-resistant parasites, and it is essential to identify the genes involved This proposal uses recently validated strategies and techniques to identify loci and genes involved in resistance to artemisinin and chloroquine, using the rodent malaria parasite, Plasmodium chabaudi. Artemisinin resistant parasites have been selected from sensitive clones. They are cloned, transmissible and are genetically stable. The chloroquine resistance (CQR) locus has been mapped within a 250 kb region of chromosome 11, syntenic with part of P. falciparum chromosome 6. This gene may be involved in CQR in P. vivax and may modulate resistance in P. falciparum. We propose to use and to further develop and optimise a validated strategy called Linkage Group Selection (LGS). The uncloned recombinant progeny of genetic crosses between drug-resistant and drug-sensitive parasites will be treated with drug to remove those parasites having the sensitive parasite?s allele at the drug resistance locus. Four quantitative molecular methods for measuring the proportions of alleles represented in mixed parasite populations can be used for the quantitative analysis of this drug treated material. Markers (from the sensitive parent) linked to the drug resistance locus will appear in reduced proportions after drug selection producing a ?selection valley? which will allow us to map the locus under selection. Backcrosses with the sensitive parent can define the locus more precisely. We will sequence the genes within the locus, using the synteny which has been demonstrated between the completed P. falciparum genome and the incomplete P. chabaudi sequence database. A sequence difference between the resistant parasite and its immediate sensitive progenitor will identify the gene. We will also attempt to identify and characterise other quantitative trait loci which play a role in high-level chloroquine resistance, by using increasing concentrations of chloroquine in LGS experiments on a genetic cross between a parasite which is resistant to a high level of chloroquine. The orthologues of all genes will be identified in P. falciparum and P. vivax. We propose to develop a sound theoretical base and statistical tools to support LGS LGS promises to hugely increase the speed with which drug resistance loci are identified, to the extent that genes will be identified even before resistant parasites are identified in the field.