Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

Abstract

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. falciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (AGC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2·5 × 10-9 mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and anti-malarial drug therapy by demonstrating that even with low mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.