Genome-wide association analysis identifies genetic loci associated with resistance to multiple antimalarials in Plasmodium falciparum from China-Myanmar border

Abstract

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine - an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.

Figure 1. In vitro parasite sensitivities to ten antimalarial drugs.

(a) IC₅₀ values to 10 different drugs and ring-stage parasite survival rates measure by RSA were sorted from the lowest to the highest values. IC₅₀ values to SP are shown in μg/ml, and RSA values are in percentage. Large discontinuous gaps were present in IC₅₀ values to SP and CQ, and ring-stage survival rates to DHA. Dashed line indicates the separation. (b) Fold change of drug sensitivities of field isolates in comparison with those of 3D7. Each column represented the sensitivities of a field isolate to 10 drugs. Hierarchical clustering of parasite isolates was analyzed using “ward” method in the R package stats. The degree of fold change is colour-coded. (c) Correlations between drug sensitivities of parasite isolates to ten antimalarial drugs. The correlations between drug sensitivities were analyzed by Spearman’s test. The degree of correlation between sensitivities of two drugs is color-coded. Drug Abbreviations: chloroquine (CQ); sulfadoxine-pyrimethamine (SP); ring-survival rates from RSA; dihydroartemisinin (DHA); artemether (AM); piperaquine (PPQ); quinine (QN); lumefantrine (LMF); and pyronaridine (PND).

Figure 2. Population structure and linkage disequilibrium in the parasite population.

(a) Population partitions identified by PCA, showing two minor outlier groups. (b) Plot of LD measured as squared correlation of allele frequencies (R²) against physical map distance (bp) between linked locus pairs in the entire population. The red solid line is the nonlinear regression trend line of R² vs. physical map distance, and dashed red line indicates R² = 0.2.

Figure 3. Manhattan plots showing the significance of SNP association in the GWAS.

Values of –log(P) for 10 drugs were plotted against chromosomal positions of SNPs. Each point represents 1 of 8572 SNPs with MAF >0.02 in a set of 94 isolates. The dashed horizontal line indicates the significance threshold of a P value of 5.83 × 10⁻⁶ after Bonferroni correction. (a) Analysis with log-transformed phenotypic data; (b) Analysis with original phenotypic data. Plots are from GEMMA, except LMF* from PLINK. The arrowheads indicate interesting SNPs. Drug Abbreviations: chloroquine (CQ); sulfadoxine-pyrimethamine (SP); ring-survival rates from RSA (DHA^RSA); dihydroartemisinin (DHA); artemether (AM); piperaquine (PPQ); quinine (QN); lumefantrine (LMF); and pyronaridine (PND).

Figure 4. Selection around resistant loci.

The SNP diversity on chromosome 4 (a₁), 7 (b₁) and 10 (c₁) are shown by the measurement of average heterozygosity of each gene, centered at pfdhfr, pfcrt and pfatg18, respectively, and the rectangle subpanel of c₁ shows the magnified view of the pfatg18 region. Red line represents SP- or CQ-resistant samples, as well as samples having the T38N mutation in pfatg18, and blue line stands for SP- or CQ-sensitive samples, or wild types in pfatg18. The disparity in diversity with grey shading likely reflects the selective sweep around resistant loci. EHH decays around pfdhfr, pfcrt and pfatg18 are shown in b₁, b₂ and b₃, with S220A in pfcrt, C59R in pfdhfr and T38N in pfatg18 as the focal SNPs. Red line indicates EHH decays in our samples, and blue line shows the decay in the reference line 3D7.

Figure 5. Plot of integrated haplotype scores (iHS) showing loci under positive selection.

SNPs with |iHS| ≥ 3.14 (top 1%) are shown above the dashed horizontal line. Plot was generated by the R package rehh.