Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2012; 109(32):13052-7. DOI: 10.1073/pnas.1210585109
Source: PubMed


Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite--the deadliest of those that cause malaria--is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites' in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.

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    • "The genomic region containing the largest number of SNPs with a high |iHS| score in Guinea is located near one end of chromosome 6, for which a similar signature was previously observed in The Gambia (Nwakanma et al. 2014) (fig. 5) as well as in Senegal (Park et al. 2012). Highly supported windows of elevated |iHS| scores were also observed on chromosomes 9 and 10, incorporating the merozoite surface protein 1 gene (msp1, PF3D7_0930300) and a cluster of different antigen genes (including GLURP, PF3D7_1035300; and msp3, PF3D7_1035400), respectively. "
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    ABSTRACT: Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year, and compared with data from 52 clinical isolates from a previously sampled population from The Gambia where there is relatively limited seasonal malaria transmission. Paired-end short read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136144 SNPs were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardised integrated haplotype scores (|iHS|) were population-specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajima's D values, including a significant over-representation of genes expressed at the merozoite stage that invades erythrocytes, and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15kb region within and flanking the gdv1 gene that regulates the early stages of gametocyte development, which is likely related to the extreme differences in mosquito vector abundance and seasonality which determine the transmission opportunities for the sexual stage of the parasite.
    Full-text · Article · Mar 2014 · Molecular Biology and Evolution
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    • "Samples were determined to be monogenomic and genetically distinct by a 24 single nucleotide polymorphism (SNP) molecular barcode [18]. Genomic DNA was sequenced using Illumina Hi-Seq machines, aligned to the 3D7 reference assembly, and variants were identified using previously described methods [19]. Using variant and invariant sites, the pfhrp2 gene coding sequence (spliced CDS sequence) was reconstructed for each sample, inserting an ‘N’ to represent an undetermined base for sites that could not be resolved due to insufficient sequence coverage. "
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    ABSTRACT: The Senegalese National Malaria Control Programme has recommended use of rapid diagnostic tests (RDTs) that target the histidine-rich protein 2 (HRP2), specific to Plasmodium falciparum, to diagnose malaria cases. The target antigen has been shown to be polymorphic, which may explain the variability in HRP2-based RDT results reported in field studies. The genetic diversity of the pfhrp2 gene has not been investigated in depth in many African countries. The goal of this study is to determine the extent of polymorphism in pfhrp2 among Senegal, Mali and Uganda parasite populations, and discuss the implications of these findings on the utility of RDTs that are based on HRP2 detection. Sequencing data from the pfhrp2 locus were used to analyze the genetic diversity of this gene among three populations, with different transmission dynamics and malaria parasite ecologies. Nucleotide diversity (pi) and non-synonymous nucleotide diversity (piNS) were studied in the pfhrp2 gene from isolates obtained in Senegal. Amino acid repeat length polymorphisms in the PfHRP2 antigen were characterized and parameters of genetic diversity, such as frequency and correlation between repeats in these populations, were assessed. The diversity survey of the pfhrp2 gene identified 29 SNPs as well as insertion and deletion polymorphisms within a 918 bp region. The Senegal pfhrp2 exhibited a substantial level of diversity [pi = 0.00559 and piNS = 0.014111 (piS = 0.0291627)], similar to several polymorphic genes, such as msp1, involved in immune responses, and the gene encoding the SURFIN polymorphic antigen, which are surface exposed parasite proteins. Extensive repeat length polymorphisms in PfHRP2, as well as similar patterns in the number, organization and the type of predicted amino acid repeats were observed among the three populations, characterized by an occurrence of Type 2, Type 4 and Type 7 repeats. These results warrant deeper monitoring of the RDT target antigen diversity and emphasize that development of other essential genes as a target for diagnostic tools is critical.
    Full-text · Article · Jan 2014 · Malaria Journal
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    • "Of the other genomic loci identified as most likely to have been under recent directional selection, a few were also identified recently from independent analysis of samples from The Gambia and Senegal using an Affymetrix custom SNP array [39] and sequence-based analysis of parasites from Senegal [38]. The selective signature on chromosome 6 was previously associated with parasites that were pyrimethamine resistant in Senegal [38], but the test could not identify the exact gene under selection. "
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    ABSTRACT: Background. Analysis of genome-wide polymorphism in many organisms has potential to identify genes under recent selection. However, data on historical allele frequency changes are rarely available for direct confirmation. Methods. We genotyped single nucleotide polymorphisms (SNPs) in 4 Plasmodium falciparum drug resistance genes in 668 archived parasite-positive blood samples of a Gambian population between 1984 and 2008. This covered a period before antimalarial resistance was detected locally, through subsequent failure of multiple drugs until introduction of artemisinin combination therapy. We separately performed genome-wide sequence analysis of 52 clinical isolates from 2008 to prospect for loci under recent directional selection. Results. Resistance alleles increased from very low frequencies, peaking in 2000 for chloroquine resistance-associated crt and mdr1 genes and at the end of the survey period for dhfr and dhps genes respectively associated with pyrimethamine and sulfadoxine resistance. Temporal changes fit a model incorporating likely selection coefficients over the period. Three of the drug resistance loci were in the top 4 regions under strong selection implicated by the genome-wide analysis. Conclusions. Genome-wide polymorphism analysis of an endemic population sample robustly identifies loci with detailed documentation of recent selection, demonstrating power to prospectively detect emerging drug resistance genes.
    Full-text · Article · Nov 2013 · The Journal of Infectious Diseases
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