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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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