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

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Social and Political Science

Abstract

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.

Technical Summary

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.

Publications

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Carter R (2007) Linkage Group Selection--a fast approach to the genetic analysis of malaria parasites. in International journal for parasitology

 
Title Comprehensive model for identifying mutations conferring resistance to antimalarial drugs 
Description 1. The development of a comprehensive and integrated model for identifying genes underlying resistance to antimalarial drugs in a rodent model malaria parasite, Plasmodium chabaudi 2. It comprises a.) the generation of isogenic drug resistant mutants by in vivo evolutionary strategies (natural selection in presence of drugs), b.) the mapping of the underlying gene mutations by genome-wide linkage analysis (Linkage Group Selection) and c.) the determination of specific mutations by whole genome re-sequencing, using the Solexa platform 3. This system is comprehensive, rapid and innovative. It has been validated by defining the single point mutations conferring resistance to artemisinin, mefloquine, lumefantrine. It has successfully specified multiple genes conferring different levels of chloroquine resitance. 4. This model can be deployed proactively to generate candidate mutations before resistance appears in the field. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact The model system as developed in this laboratory has serious public health implications. It will proactively generate gene candidates before resistance arises in the field even when no information is available regarding mode of drug action or mode of resistance. Additionally, it has evolutionary impacts, regarding the genetic architecture of naturally selected determinants of resistance phenotypes 
 
Title Pyrosequencing (allele quantitation) 
Description We have developed a comprehensive genome-wide library of mapped quantitative SNP allele assays. They allow the genome-wide mapping of gene mutations underlying selectable phenotypes, such as drug resistance and strain-specific immunity 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The mapping of the genetic determinants of medically important traits (drug resistance and strain-specific immunity) 
 
Title Quantitative whole genome sequencing 
Description Identification of genome-wide SNPs between parental strains. Their proportions can be measured in the un-selected and drug-selected recombinant progeny of genetic crosses. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Unknown 
 
Title Statistical analysis 
Description We have developed two independent methods for evaluating the statistical significance of areas where alleles from the sensitive parent are under-represented (selection valleys) in the progeny of parasites (after drug treatment compared to no treatment). 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact This confers confidence in assigning particular areas of the genome to particular selectable phenotypes. 
 
Description Artemisinin resistance in Plasmodium falciparum (laboratory) 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution Information regarding role of specific gene mutations conferring resistance to artemisinin in Plasmodium chabaudi
Collaborator Contribution Evaluation of genetic mutations conferring artemisinin resistance (in Plasmodium chabaudi) by transfection of Plasmodium falciparumEvaluation of additional genetic markers of artemisinin resistance by transfection in Plasmodium falciparum
Impact Evaluation of the possible role of ubp1 (gene) in in vitro selected artemisinin resistant mutants of Plasmodium falciparum (PubMed ID 17581118), and ongoing research
Start Year 2007
 
Description Artemisinin resistance in Plasmodium falciparum (laboratory) 
Organisation Nagasaki University
Country Japan 
Sector Academic/University 
PI Contribution Information regarding role of specific gene mutations conferring resistance to artemisinin in Plasmodium chabaudi
Collaborator Contribution Evaluation of genetic mutations conferring artemisinin resistance (in Plasmodium chabaudi) by transfection of Plasmodium falciparumEvaluation of additional genetic markers of artemisinin resistance by transfection in Plasmodium falciparum
Impact Evaluation of the possible role of ubp1 (gene) in in vitro selected artemisinin resistant mutants of Plasmodium falciparum (PubMed ID 17581118), and ongoing research
Start Year 2007
 
Description Genetic determination of resistance to multiple anti-malarial drugs 
Organisation University of Lisbon
Country Portugal 
Sector Academic/University 
PI Contribution We provided the leadership, concepts, resources, expertise, materials, equipment and facilities required for the phenotyping, genetic and genomic analysis of drug-resistant mutants and the identification of the mutations conferring these phenotypes
Collaborator Contribution Supply of materials (mutant clones), knowledge, ideas etc Collaborative visits of PhD students to undertake studies regarding the phenotype, genetics and genomics of drug resistance in Plasmodium chabaudi Assistance with the analysis of whole genome re-sequencing of drug resistant mutants
Impact Generation and characterisation of drug-resistant mutants using rodent malaria parasites, Plasmodium chabaudi Genetic analysis of these mutants and development of relevant resources Genome resequencing and identification of critical mutations underlying resistance to chloroquine, artemisinin, mefloquine and lumefantrine (PubMed 16437600, 17222845, 17581118) and publications in submission.
 
Description Malaria Nexus Interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Extended, in depth interview for Elsevier based 'Malaria Nexus'

Unknown
Year(s) Of Engagement Activity 2011
 
Description Media Coverage - Artemisinin UBP1 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Extensive local, national and international coverage of research findings in BMC Genomics. Included all major Scottish dailies, BBC Scotland live interview, Radio Forth etc

Invitations to teach on MSc courses (University of Edinburgh)
Year(s) Of Engagement Activity 2010
 
Description Podcast EUSci 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Interview for University Magazine

Unknown
Year(s) Of Engagement Activity 2010
 
Description Podcast SBS 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Description of the genetic changes causing drug resistance in malaria parasites

Not known
Year(s) Of Engagement Activity 2007