Multiple genetic backgrounds of the amplified Plasmodium falciparum multidrug resistance (pfmdr1) gene and selective sweep of 184F mutation in Cambodia

Abstract

Background: The emergence of artesunate-mefloquine (AS+MQ)-resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia.

Methods: We characterized 13 microsatellite loci flanking (+/-99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene.

Results: Genetic analysis revealed no difference in the mean (+/- standard deviation) expected heterozygosity (H(e)) at loci around single (0.75+/-0.03) and multiple (0.76+/-0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean H(e) (+/-SD) around mutant allele (0.56+/-0.05), compared with wild-type allele (0.84+/-0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele.

Conclusion: The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.

Conflict of interest statement

Potential conflicts of interest: none reported.

Figures

Figure 1

A map of Cambodia showing the location of the 4 sites (Pailin, Kampong Seila, Memut, and Rattanakiri) from which the isolates used in this study were obtained. The proportion of mefloquine (MQ) resistance in Cambodia and bordering areas (Thailand to the west, Vietnam to the east, and Laos to the north) is indicated by pie charts.

Figure 2

The expected heterozygosity (He) at 13 microsatellite loci around pfmdr1. Comparison of He between groups of isolates with a single copy or multiple copies of pfmdr1. The mean He at neutral loci on chromosomes 2 and 3 is shown by a dotted line. The error bars indicate ± 1 standard deviation. A, isolates from both western and eastern Cambodia; B, detail showing the closest 8 microsatellite loci flanking (± 9 kb) pfmdr1 for isolates from western and eastern Cambodia; C, isolates from western Cambodia; D, isolates from eastern Cambodia.

Figure 3

The expected heterozygosity (He) at 13 loci around wild-type (184Y) and mutant (184F) pfmdr1. Comparison of He between 184Y and 184F pfmdr1 groups of isolates from both western and eastern Cambodia (A) and the closest 8 microsatellite loci flanking (± 9 kb) pfmdr1 (B), and comparison of 184F group of isolates from western Cambodia with 184F and 184Y groups of isolates from eastern Cambodia (C) and the closest 8 microsatellite loci flanking (± 9 kb) pfmdr1 (D). The mean He at neutral loci on chromosomes 2 and 3 is shown by a dotted line. The error bars indicate ± 1 standard deviation.

Figure 4

Pair-wise linkage disequilibrium around pfmdr1 (± 9 kb). A, linkage disequilibrium between loci within the wild-type group (lower half of the diagonal) and mutant group (upper half of the diagonal). B, linkage disequilibrium between loci within the group of isolates with a single copy of pfmdr1 (lower half of the diagonal) and those with multiple copies of pfmdr1 (upper half of the diagonal). Each cell shows a comparison between polymorphic pairs of loci. P < .006 was considered to be significant after Bonferroni correction. Light gray cells represent P values that are not significant (>.006), and dark gray cells indicate statistically significant P values (<.006).

Figure 5

Median-joining network showing genetic relationships among pfmdr1 haplotypes in Cambodia. The closest 8 loci (± 9 kb) were used for constructing the network. Each circle in this network represents a unique haplotype, with the size of the circle being proportional to the number of isolates harboring that haplotype. The circles shown in yellow and gray represent isolates carrying single and multiple copies of pfmdr1, respectively. The haplotype shared by both isolates with single and isolates with multiple pfmdr1 copies (proportion indicated in pie charts) are shown as a circle with both yellow and gray shading. The red dots are the median vectors that imply software-generated “hypothetical haplotypes,” joining 2 haplotypes in the diagram from which single and/or multiple pfmdr1 copy–containing haplotypes may have originated. The isolates with 184F are marked by blue arrows enclosed in the shaded clusters.