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Adaptive Copy Number Evolution in Malaria Parasites

17 Nov 2008

Marcia Triunfol

Source: PLoS Genetics (see original article)

Citation: Nair S, Miller B, Barends M, Jaidee A, Patel J, et al. (2008). Adaptive Copy Number Evolution in Malaria Parasites. PLoS Genet 4(10): e1000243.

Genetics taught in high school says that individuals of the same species are expected to share not only the same genes that are characteristic of the species, but also the same copy number of such genes. Well, not so fast!

Genome sequencing of human DNA from different individuals keeps surprising us with unexpected, and sometimes unexplained, findings. One such finding is the discovery that humans can have different copies of specific genes. For instance, a recent study showed that populations that eat a lot of starch have more copies of the amylase gene, which is the gene that produces the amylase protein found in the mouth cells and which helps degrade starch. In other words, those who eat more starch need more amylase to degrade it, and therefore have an increased copy number of the gene that carries the code to produce the amylase protein. This phenomenon is known as copy number variation (CNP) and it has also been observed in other vertebrates.

Like the human genome, the malaria parasite genome has many CNPs. One such CNP found in chromosome 5 is known to be related to drug resistance because manipulation of its copy number results in different responses to multiple drugs. Thus, it is possible that copy number variation in other genes of the plasmodium’s genome is related to drug resistance as well.

One such possibility is the copy number variation observed in a gene known as GTP-cyclohydrolase I (gchI), which encodes the first enzyme of the plasmodium folate biosynthesis pathway. This pathway includes five more enzymes that act in cascade and which together are responsible for metabolizing folate. Two of these five enzymes, dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), have been the target of antimalarial drugs. However, genetic mutations in these two enzymes have already generated strains with drug resistance, even though such strains show reduced folate metabolism.

Is it then possible that the observed increase in the gchI copy number is a way of compensating for the reduced efficiency of other enzymes located downstream in the folate pathway biosynthesis? This is the main hypothesis that a study by Nair and colleagues aimed to test.

To do so, the researchers examined the genetic population of the Plasmodium falciparum in two neighbouring countries, Laos and Thailand. The two countries were chosen because they have different histories with using antifolate drugs. While anti-folate drugs were heavily used in Thailand (which led to selection strains with drug resistance), in Laos these drugs were not used frequently.

Using molecular biology techniques capable of detecting gene copy numbers in a single strain, the authors observed that plasmodium strains from Thailand showed numbers of copies of the gchI gene that varied between 1 and 11 per genome, while in Laos most strains had only one copy, with very few presenting two copies of the gchI gene. Interestingly, samples collected in Thailand had widespread mutations in both dhfr and dhps genes, with predominance (80%) of the dhfr-164L mutation that is known to impair folate metabolism. In Laos, this same mutation was not found in any strain and most parasites carried non-mutated (wild type) copies of the dhps gene.

If the gch CNP is indeed a way of compensating for less efficient folate pathway enzymes selected in response to antimalarial drugs used in Thailand in the 70s and 80s, then this CNP represents a genetic event that took place very recently in history and therefore genetic diversity among these copies should be low, as not enough time has passed to fix genetic changes. The analysis of genetic markers located in the flanking areas and vicinity of gchI confirmed that genetic diversity in samples from Thailand was much lower than that observed in samples from Laos. This finding supports the hypothesis that gch CNP is related to the presence of mutations in the enzymes of the folate pathway.

Next, the researchers observed that strains carrying the dhfr-164L mutation (known to cause high level resistance to antifolate drugs) had a particularly high copy number of gch, if compared to strains carrying other mutations in the folate pathway enzymes – i.e, in strains carrying a extremely poor copy of the dhfr gene, the copy number of gch was even higher.

Together, these findings show that the pressure caused by antimalarial drugs can trigger genetic solutions not previously known and that multiple enzymes in the drug-target pathway may be affected, and then adapted, as a way to compensate for less effective enzymes.

© 2008 Nair et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Source

Title:
Adaptive copy number evolution in malaria parasites.

Authors:
Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ

References:
PLoS Genet. Oct 2008: Vol. 4 no. 10, pp. e1000243

Abstract:
Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

PMID: 18974876
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