A major genome region underlying artemisinin resistance in malaria

Abstract

Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10(-6) to 10(-12)) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.

Figure 1

Phenotypic and genetic differentiation between South-East Asian parasites. A) Patterns of parasite clearance from two Thai patients (black, slow CR/red fast CR) following treatment. B) F_ST between locations for 5,662 SNPs with MAF > 5%. C) Clearance half lives from Laos (blue; n=44), Cambodia (red; n=64), and Thailand (black; n=3,202) and D) PCA of parasites using 1,770 SNPs with MAF >5% and no missing data.

Figure 2

Evidence for selection. Plots show F_ST and XP-EHH for each SNP (n=6,969) in pair-wise comparisons of countries. Dashed lines represent the 1% level from the genomic empirical distribution. Significant windows are shown by black bars above each plot. Red = Cambodia vs. Laos, blue = Thailand vs. Laos, black = Cambodia vs. Thailand. A) Chromosome 1. No evidence of selection surrounding atpase6. B) Patterns of selection surrounding five known drug resistance genes (marked by arrows). C) Three loci putatively under selection.

Figure 3

Association testing. A) Study sites on the Thai-Burma border. Northern populations were Maela Camp and Wang Pha and southern populations Mae Kong Khen and Mawker Thai. Clearance rate between 2001-2010 has declined (shown above the map). The y-axis shows the percentage of parasites with –log(clearance rate) > 1.89. B-D) Association of 94 SNPs from selected regions (black) and 93 genome-wide SNPs (red). Two neighboring SNPs in the northern population from 2007-2010 showed strong association with clearance rate (denoted 1 and 2). E) Boxplot of the clearance rate for each allele of SNP 1. F) The chromosomal context surrounding these SNPs.

Figure 4

Fine mapping using 19 microsatellites. A) Association P-values from the early (2001-4; red dots) and late (2007-10; black dots) periods in northern Thai population. Bonferroni-correction threshold is shown by dashed line. B) Comparison of H_e in Thailand, Laos and Cambodia. C) EHH surrounding a microsatellite (position 1,763,950) in slow (half-life>4.6 hours) and fast (half-life<2.3 hours) clearing parasites. D) Phenotypic distribution at this locus in 2007-10 (P=4×10^-12).