Are transporter genes other than the chloroquine resistance locus (pfcrt) and multidrug resistance gene (pfmdr) associated with antimalarial drug resistance?

Abstract

Mu et al. (Mu, J., M. T. Ferdig, X. Feng, D. A. Joy, J. Duan, T. Furuya, G. Subramanian, L. Aravind, R. A. Cooper, J. C. Wootton, M. Xiong, and X. Z. Su, Mol. Microbiol. 49:977-989, 2003) recently reported exciting associations between nine new candidate transporter genes and in vitro resistance to chloroquine (CQ) and quinine (QN), with six of these loci showing association with CQ or QN in a southeast Asian population sample. We replicated and extended this work by examining polymorphisms in these genes and in vitro resistance to eight drugs in parasites collected from the Thailand-Burma border. To minimize problems of multiple testing, we used a two-phase study design, while to minimize problems caused by population structure, we analyzed parasite isolates collected from a single clinic. We first examined associations between genotype and drug response in 108 unique single-clone parasite isolates. We found strong associations between single nucleotide polymorphisms in pfmdr and mefloquine (MFQ), artesunate (AS), and lumefantrine (LUM) response. We also observed associations between an ABC transporter (G7) and response to QN and AS and between another ABC transporter (G49) and response to dihydro-artemisinin (DHA). We reexamined significant associations in an independent sample of 199 unique single-clone infections from the same location. The significant associations with pfmdr-1042 detected in the first survey remained. However, with the exception of the G7-artesunate association, all other associations observed with the nine new candidate transporters disappeared. We also examined linkage disequilibrium (LD) between markers and phenotypic correlations between drug responses. We found minimal LD between genes. Furthermore, we found no correlation between chloroquine and quinine responses, although we did find expected strong correlations between MFQ, QN, AS, DHA, and LUM. To conclude, we found no evidence for an association between 8/9 candidate genes and response to eight different antimalarial drugs. However, the consistent association observed between a 3-bp indel in G7 and AS response merits further investigation.

FIG. 1.

(A) Box-and-whisker plots showing associations between polymorphisms and drug response in phase I samples. (B) The same associations examined in phase II samples. Asterisks above graphs indicate significance level (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The boxes indicate the 25th and 75th percentiles, the whiskers indicate the 10th and 90th percentiles, and the lines show the median values. The numbers at the top of each graph indicate the sample sizes for each of the alternate states at the polymorphism examined. Only the pfmdr-1042 and G7 polymorphisms remain significantly associated with drug response in both phase I and II population samples examined.

FIG. 2.

Scatter plots showing phenotypic correlations between log (IC₅₀) values for different drugs. (A) CQ and QN; (B) DHA and AS; (C) MFQ and QN; (D) LUM and MFQ. Correlation coefficients (r²), sample sizes, and significance for all pairwise comparisons are shown in Table 4.

FIG. 3.

Linkage disequilibrium between 15 polymorphic markers (SNPs or microsatellites) in 10 transporter genes. Panel A shows results from this study. Panel B shows results from Mu et al.'s Southeast Asian data set for comparison (14). Significance was assessed by permutation as described in the text (black shading, P < 0.01; grey shading, P < 0.05). Significant comparisons between markers located within the same gene are marked with a white “w” to distinguish them from between-gene comparisons.