Characteristics of genetic hitchhiking around dihydrofolate reductase gene associated with pyrimethamine resistance in Plasmodium falciparum isolates from India

Abstract

Sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum has been widespread across continents, causing the major hurdle of controlling malaria. Resistance is encoded mainly by point mutations in P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) target genes. To study the origin and evolution of pyrimethamine resistance on the Indian subcontinent, microsatellite markers flanking the pfdhfr gene were mapped. Here we describe the characteristics of genetic hitchhiking around the pfdhfr gene among 190 P. falciparum isolates. These isolates were collected from five different geographical regions of India (Uttar Pradesh, Madhya Pradesh, Assam, Orissa, and Andaman and Nicobar Islands) where malarial transmission rates and levels of drug resistance vary across regions. Among the isolates, we observed a significant reduction in genetic variation in the +/-20-kb vicinity of the mutant pfdhfr alleles due to hitchhiking. This reduction in genetic diversity was more prominent around quadruple pfdhfr alleles (heterozygosity [H(e)] = 0.23) than around double (H(e) = 0.365) and single (H(e) = 0.465) mutant alleles. Asymmetry in the selective sweep flanking the pfdhfr alleles was observed with regional isolates, emphasizing the drug usage with the parasite population. All the pfdhfr alleles share a single microsatellite haplotype and seem to have originated from a single progenitor similar to that of Southeast Asian (Thailand) pfdhfr mutants. Results of the present study also indicate that the emergence of drug-resistant alleles is a recent phenomenon in India compared to Southeast Asian countries.

FIG. 1.

Map of India showing sample collection sites.

FIG. 2.

Genetic variations around the pfdhfr gene among different alleles. Microsatellite markers along the x axis are plotted against the expected H_e values. H_e values of Indian P. falciparum isolates having different pfdhfr alleles are shown as the following: □, A₁₆N₅₁C₅₉S₁₀₈I₁₆₄; ▵, A₁₆N₅₁C₅₉N₁₀₈I₁₆₄; ⋄, A₁₆N₅₁R₅₉N₁₀₈I₁₆₄; ○, A₁₆I₅₁R₅₉N₁₀₈I₁₆₄/A₁₆N₅₁R₅₉N₁₀₈L₁₆₄; ×, A₁₆I₅₁R₅₉N₁₀₈L₁₆₄.

FIG. 3.

Genetic variations around the pfdhfr gene among different geographical regions. Microsatellite markers along the x axis are plotted against the expected H_e values. H_e values of mutant A₁₆N₅₁C₅₉N₁₀₈I₁₆₄ (a), A₁₆N₅₁R₅₉N₁₀₈I₁₆₄ (b), A₁₆I₅₁R₅₉N₁₀₈I₁₆₄/A₁₆N₅₁R₅₉N₁₀₈L₁₆₄ (c), and A₁₆I₅₁R₅₉N₁₀₈L₁₆₄ (d) pfdhfr alleles from UP (▴), MP (•), Assam (♦), Orissa (▾), and Andaman and Nicobar Islands (*) are shown. H_e values of isolates having the wild-type A₁₆N₅₁C₅₉S₁₀₈I₁₆ pfdhfr allele are shown as ▪.

FIG. 4.

Decline in linkage around the pfdhfr gene (x axis denotes the distance of the microsatellite marker from the pfdhfr gene [in kb], whereas the y axis denotes the value of EHH at that locus). □, A₁₆N₅₁C₅₉S₁₀₈I₁₆₄; ▵, A₁₆N₅₁C₅₉N₁₀₈I₁₆₄; ⋄, A₁₆N₅₁R₅₉N₁₀₈I₁₆₄; ○, A₁₆I₅₁R₅₉N₁₀₈I₁₆₄/A₁₆N₅₁R₅₉N₁₀₈L₁₆₄; *, A₁₆I₅₁R₅₉N₁₀₈L₁₆₄.