Wide variation in microsatellite sequences within each Pfcrt mutant haplotype

Abstract

Flanking microsatellites for each of the Pfcrt mutant haplotype of Plasmodium falciparum remain conserved among geographical isolates. We describe here heterogeneity in the intragenic microsatellites among each of the Pfcrt haplotype. There were fourteen different alleles of AT repeats of intron 2 and eight alleles of TA repeats of intron 4 of the pfcrt gene among Indian isolates. This resulted in 33 different two-locus (intron 2 plus intron 4) microsatellite genotypes among 224 isolates. There were 15 different two-locus microsatellite genotypes within the South American Pfcrt haplotype (S₇₂V₇₃M₇₄N₇₅T₇₆S₂₂₀) and 11 genotypes in the southeast Asian haplotype (C₇₂V₇₃I₇₄E₇₅T₇₆S₂₂₀) in these isolates. Indian isolates with Pfcrt haplotype C₇₂V₇₃I₇₄E₇₅T₇₆S₂₂₀ shared one of its two-locus microsatellite genotype with southeast Asian P. falciparum parasite lines from Thailand (K1) and Indochina (Dd2 and W2). Conversely, Indian isolates containing S₇₂V₇₃M₇₄N₇₅T₇₆S₂₂₀ Pfcrt haplotype did not share any of their two-locus microsatellite genotype with South American parasite line 7G8 from Brazil. Significantly, large number of newer two-locus microsatellite genotypes were detected in a 2-year time period (P < 0.05). Microsatellite variation was more prominent in the areas of high malaria transmission. It is concluded that the genetic recombination in the intragenic microsatellites continues in the parasite population even after microsatellites flanking the pfcrt gene had already been fixed. Presence of various Pfcrt haplotypes and a variety of intragenic microsatellites indicates that there is a wide spectrum of chloroquine resistant parasite population in India. This information should be useful for malaria control programs of the country.

Introduction

Plasmodium falciparum, cause of the most malignant form of malaria, has developed resistance towards the most commonly used antimalarial drug chloroquine. Although it took a longer time to develop resistance against this drug, its spread to other countries had been faster [1]. Payne proposed two independent origins of chloroquine resistance [1]. However, molecular studies revealed at least four independent foci of chloroquine resistance, i.e. one in southeast Asia which had spread to Africa, one in Papua New Guinea, and two in South America [2], [3], [4]. Presence of additional origins of chloroquine resistance have also been proposed in Philippines and Cambodia [5], [6], [7].

Microsatellite or simple sequence repeats (SSRs) are abundant in the P. falciparum genome [8]. P. falciparum microsatellite markers had been used to study population genetics and evolutionary biology of this parasite. They had also been used to trace the origin and spread of drug resistance through the continents [7], [9]. Increase in drug pressure to parasite population results in occurrence and subsequent rapid spread of new mutations for survival of the parasite. This rapid spread of drug resistant mutations influences the frequency of flanking microsatellite sequences. Recombinational events for spread of drug resistant mutations occur between sites that are far apart and therefore the closest flanking microsatellite markers remain in linkage disequilibrium with the resistant allele [10]. Therefore, it had been proposed that microsatellite markers should be positioned as close as possible to the drug resistance gene since P. falciparum shows a high rate of recombination [11]. Markers positioned within 5 kb range are proposed to be ideal although recombination events may affect this as well [10]. Microsatellites positioned in the closest flanking regions of the chloroquine resistance marker gene have been found to be fixed among the parasite population of southeast Asia, South America, and Papua New Guinea which have a distinctive pattern of point mutations in the coding region of the pfcrt gene [2], [3], [4]. Tracing the resistant parasite population and its evolution is important in designing the malaria control strategies. Although microsatellites are present within the pfcrt gene, they have not been investigated extensively for evolutionary purpose [5]. We describe here the sequence analysis of two microsatellites present in the downstream introns of exon 2 and 4 among various pfcrt mutant haplotypes present in Indian isolates. We found multiple intragenic microsatellite alleles among each of the pfcrt mutant haplotype.