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Researchers Find Gene Associated with Artemisinin Resistance in Malaria

Image courtesy of the CDC.


What was the research project?
A 2013 Nature News & Views article covered the main points of the Ariey et al., 2013 research project, which sought to determine the genetic cause of artemisinin-resistant malaria (Plowe, 2013; Ariey et al., 2013). Malaria in humans is caused by parasites of the genus Plasmodium, but most infections are specifically caused by Plasmodium falciparum (Nadjm and Behrens, 2012). Artemisinin (ART) is a drug that is the standard treatment for malaria and it typically cures patients completely after two days of proper dosages. However, many P. falciparum infections in southeast Asia, particularly in Cambodia, are becoming more difficult or impossible to treat because the parasite has developed ART-resistance.

To promote detection and control ART-resistant malaria, Ariey et al. conducted a genome-wide association study (GWAS) of P. falciparum strains to determine a molecular marker for ART-resistance. Specifically, Ariey and colleagues isolated an ART-sensitive P. falciparum strain from Tanzania and exposed it to small doses of ART derivatives over 125 cycles to develop an ART-resistant strain. Their GWAS compared their ART-resistant strain, the original ART-sensitive strain from Tanzania, and a known ART-resistant strain from Cambodia at various time points over the course of 125 drug-pressure cycles. Genes in ART-resistant strains with single nucleotide polymorphisms (SNPs) not present in the ART-sensitive strain became candidate ART-resistance genes. The unique SNPs were further analyzed through determining in vitro parasite survival rates when strains are exposed to ART and in vivo parasite clearance rates when infected patients took ART treatments. The researchers ultimately determined that mutations in a kelch-propeller (K13-propeller) domain are associated with ART-resistance (Figure 1).



Figure 1. Kelch protein motif. Kelch proteins, like the kelch 13 protein, form a pinwheel tertiary structure.
The propeller-like domains, made of amino-acid sequence repeats, are used in protein-protein interactions.
Image courtesy of Wikimedia Commons.


Were they testing a hypothesis or doing discovery science?
Ariey et al. were doing discovery science. They began their GWAS without hypothesized candidate ART-resistance genes. They used genome comparisons to reveal unique SNPs in ART-resistant strains and genes with these unique SNPs then became candidate genes for further in vitro and in vivo analysis. 


What genomic technology was used in the project?
The researchers used Illumina technology for whole-genome sequencing to compare the P. falciparum strains at various time points. The sequencing reads were filtered using a software that trims the end low-quality bases in individual reads. The trimmed reads were then aligned to generate a full contig, which is a sequence made up of overlapping consensus reads, for each strain. The researchers also PCR amplified candidate genes and sequenced the PCR products to confirm the presence of unique SNPs.


What was the take home message?
Mutations in a gene coding for the K13-propeller protein are associated with P. falciparum ART-resistance. Therefore, the reported mutations in the K13 gene can be
tentatively used as molecular markers to detect ART-resistant strains and prevent their spread. This project established the groundwork for future studies to continue characterizing K13 mutants and mapping the marker throughout southeast Asia.


What is your evaluation of the project?
I appreciated that the researchers developed their own ART-resistant strain from a naturally occurring ART-sensitive strain. The ART-sensitive strain was able to be a control for their "in-house" ART-resistant strain to account for SNPs present in that specific isolated strain that are not associated with ART-resistance. Therefore, having this control for background mutations allowed them to narrow in on candidate SNPs much more effectively. I also thought that this study was a great example of how genomic research is not constrained to only hypothesis-testing studies. Rather, this study was discovery-based and hypothesis-generating. The researchers did not begin the study with putative ART-resistance genes; they used genome comparisons to hone in on candidate genes and ultimately found at least one of the possible multiple ART-resistance molecular markers. These finding are hypothesis-generating as well because they will fuel further genetic research to determine if K13 mutations are alone sufficient to confer ART-resistance or if more genes are involved. I thought that this study was a great use of genomic technology to answer an important and seemingly overwhelming question of what mutations are associated with ART-resistance.


References

Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P, Leang R, Duong S, Sreng, S, Suon S, Chuor CM, Bout DM, Menard S, Rogers WO, Genton B, Fandeur T, Miotto O, Ringwald P, Bras JL, Berry A, Barale JC, Fairhurst RM, Benoit-Vical F, Mercereau-Puijalon O, Menard D. 2013. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 505(7481): 50-55.

Nadjm B, Behrens RH. 2012. Malaria: an update for physicians. Infect Dis Clin North Am. 26(2): 243-259.


Plowe CV. 2013. Malaria: Resistance nailed. Nature. 505(7481): 30-31.


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