Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity

Abstract

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine.

Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset.

Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity.

Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Keywords: Plasmodium; chloroquine resistance transporter; falciparum; malaria; piperaquine; plasmepsin; resistance.

Figure 1.

Distribution of piperaquine 90% inhibitory concentrations (IC₉₀) by genetic subpopulation. Parasites in the genome-wide analysis grouped into 6 genetic subpopulations. Most parasites with elevated piperaquine IC₉₀s grouped together in genetic subpopulation 2 (n = 22). Abbreviations: IC₉₀, 90% inhibitory concentration; PPQ, piperaquine.

Figure 2.

Manhattan plot of the association between genome-wide parasite genotypes and piperaquine 90% inhibitory concentrations (IC₉₀). Linear mixed models were used to estimate the association between single nucleotide polymorphisms (SNPs) on the DNA microarray and a continuous measure of log-transformed piperaquine IC₉₀ (n = 153). The SNPs achieving genome-wide significance are shown in red. The SNPs on chromosomes 6, 10, 11, 12, and 14 were significantly associated with the phenotype. The SNP in PfCRT encoding the F145I mutation was not included in the microarray and was only detected by whole-genome sequencing.

Figure 3.

Random forest variable importance plot identifying loci from the DNA microarray that best predict piperaquine 90% inhibitory concentrations (IC₉₀). Random forests were used to identify loci that were the best predictors of a continuous measure of log-transformed piperaquine IC₉₀ in samples from genetic subpopulation 2 (n = 22). The single nucleotide polymorphisms (SNPs) showing the greatest percentage increase in mean squared error (x-axis) are the best predictors of the phenotype. The SNPs on chromosomes 7, 8, 9, 10, 11, and 14 were the top predictors of log-transformed IC₉₀.

Figure 4.

Association between gene copy number and piperaquine 90% inhibitory concentrations (IC₉₀). Linear regression was used to model normalized coverage of Plasmodium falciparum core genes in 117 whole-genome sequences as a function of log-transformed piperaquine IC₉₀ (treated as a continuous variable)_. Normalized coverage of the plasmepsin II/III genes showed the strongest association with piperaquine IC₉₀ (P values indicated in red).

Figure 5.

Distribution of piperaquine 90% inhibitory concentrations (IC₉₀) among parasites with different combinations of amplified plasmepsin II copy number and PfCRT 145I. Box plots indicate the distribution of piperaquine IC₉₀ in parasites from the validation data set (n = 53) in the following groups: parasites with a single copy of plasmepsin II and without the PfCRT 145I mutation (left), parasites with >1 copy of plasmepsin II and without PfCRT 145I (center), and parasites with >1 copy of plasmepsin II and with PfCRT 145I (right). The PfCRT 145I mutation only occurred in parasites with amplified plasmepsin II copy number. Abbreviation: IC₉₀, 90% inhibitory concentration.