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Adaptive copy number evolution in malaria parasites.

Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ - PLoS Genet. (2008)

Bottom Line: The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP.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.

View Article: PubMed Central - PubMed

Affiliation: Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX, USA.

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.

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The dhfr-164L resistance mutation is associated with gch1 CNP in Thailand.(a) Thai parasites were ranked by gch1 CNP. Error bars show 95% CI around copy number estimates. Open black circles or closed red circles indicate isolates carrying 164I or 164L at dhfr. (b) Association between 55 polymorphic sSNPs and gch1 CNP. Raw p-values from t-tests of log transformed copy number estimates are plotted; very similar p-values were obtained from non-parametic Mann-Whitney U-tests using untransformed copy number estimates. Dotted horizontal lines show thresholds for significance following Bonferroni correction for multiple testing. The bars show the strength of associations between gch1 CNP and dhfr-I164L (solid red), between polymorphic SNPs in dhfr (N51I) and dhps (S436A, K540E, A581G) (open red bars), and between 55 sSNPs (black). There was only one SNP (MAL04-469608) other than dhfr-164L that crossed the Bonferroni-corrected threshold of p<0.05.
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pgen-1000243-g006: The dhfr-164L resistance mutation is associated with gch1 CNP in Thailand.(a) Thai parasites were ranked by gch1 CNP. Error bars show 95% CI around copy number estimates. Open black circles or closed red circles indicate isolates carrying 164I or 164L at dhfr. (b) Association between 55 polymorphic sSNPs and gch1 CNP. Raw p-values from t-tests of log transformed copy number estimates are plotted; very similar p-values were obtained from non-parametic Mann-Whitney U-tests using untransformed copy number estimates. Dotted horizontal lines show thresholds for significance following Bonferroni correction for multiple testing. The bars show the strength of associations between gch1 CNP and dhfr-I164L (solid red), between polymorphic SNPs in dhfr (N51I) and dhps (S436A, K540E, A581G) (open red bars), and between 55 sSNPs (black). There was only one SNP (MAL04-469608) other than dhfr-164L that crossed the Bonferroni-corrected threshold of p<0.05.

Mentions: The demonstration that a derived gch1 CNP has rapidly spread to high frequency within Thai parasite populations, but not in neighboring populations from Laos, provides strong evidence that gch1 is adaptive, but provides few clues about the nature of the selection involved. We reasoned that if gch1 CNP is involved directly or indirectly in resistance to antifolate drugs, we might expect to see genetic evidence for interactions with genes involved in resistance downstream in the folate biosynthesis pathway. We therefore examined associations between gch1 copy number and known mutations that underlie antifolate resistance in dhfr (chr. 4) and dhps (chr. 8). In Thailand parasites bearing dhfr-164L carried significantly higher copy number of gch1 (t = −4.313, p = 0.000026) than those bearing dhfr-164I (Figure 6a). More marginal associations were also observed for dhfr-N51I (t = −1.964, p = 0.051) and two sites (A436S and A581G) in dhps (t = −2.184, p = 0.033) in perfect LD. To empirically test the significance of the dhfr-164L result we compared associations between gch1 copy number and 55 sSNPs with minor allele frequency >5% in Thailand. The dhfr-164L association was the strongest observed and remained significant after correction for multiple tests, arguing that this association cannot be explained by population structure (Figure 6b). These results reveal the genetic signature of functional interaction (epistasis for fitness) between two physically unlinked genes in the same biochemical pathway, providing strong evidence that gch1 CNP results either directly or indirectly from antifolate selection.


Adaptive copy number evolution in malaria parasites.

Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ - PLoS Genet. (2008)

The dhfr-164L resistance mutation is associated with gch1 CNP in Thailand.(a) Thai parasites were ranked by gch1 CNP. Error bars show 95% CI around copy number estimates. Open black circles or closed red circles indicate isolates carrying 164I or 164L at dhfr. (b) Association between 55 polymorphic sSNPs and gch1 CNP. Raw p-values from t-tests of log transformed copy number estimates are plotted; very similar p-values were obtained from non-parametic Mann-Whitney U-tests using untransformed copy number estimates. Dotted horizontal lines show thresholds for significance following Bonferroni correction for multiple testing. The bars show the strength of associations between gch1 CNP and dhfr-I164L (solid red), between polymorphic SNPs in dhfr (N51I) and dhps (S436A, K540E, A581G) (open red bars), and between 55 sSNPs (black). There was only one SNP (MAL04-469608) other than dhfr-164L that crossed the Bonferroni-corrected threshold of p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570623&req=5

pgen-1000243-g006: The dhfr-164L resistance mutation is associated with gch1 CNP in Thailand.(a) Thai parasites were ranked by gch1 CNP. Error bars show 95% CI around copy number estimates. Open black circles or closed red circles indicate isolates carrying 164I or 164L at dhfr. (b) Association between 55 polymorphic sSNPs and gch1 CNP. Raw p-values from t-tests of log transformed copy number estimates are plotted; very similar p-values were obtained from non-parametic Mann-Whitney U-tests using untransformed copy number estimates. Dotted horizontal lines show thresholds for significance following Bonferroni correction for multiple testing. The bars show the strength of associations between gch1 CNP and dhfr-I164L (solid red), between polymorphic SNPs in dhfr (N51I) and dhps (S436A, K540E, A581G) (open red bars), and between 55 sSNPs (black). There was only one SNP (MAL04-469608) other than dhfr-164L that crossed the Bonferroni-corrected threshold of p<0.05.
Mentions: The demonstration that a derived gch1 CNP has rapidly spread to high frequency within Thai parasite populations, but not in neighboring populations from Laos, provides strong evidence that gch1 is adaptive, but provides few clues about the nature of the selection involved. We reasoned that if gch1 CNP is involved directly or indirectly in resistance to antifolate drugs, we might expect to see genetic evidence for interactions with genes involved in resistance downstream in the folate biosynthesis pathway. We therefore examined associations between gch1 copy number and known mutations that underlie antifolate resistance in dhfr (chr. 4) and dhps (chr. 8). In Thailand parasites bearing dhfr-164L carried significantly higher copy number of gch1 (t = −4.313, p = 0.000026) than those bearing dhfr-164I (Figure 6a). More marginal associations were also observed for dhfr-N51I (t = −1.964, p = 0.051) and two sites (A436S and A581G) in dhps (t = −2.184, p = 0.033) in perfect LD. To empirically test the significance of the dhfr-164L result we compared associations between gch1 copy number and 55 sSNPs with minor allele frequency >5% in Thailand. The dhfr-164L association was the strongest observed and remained significant after correction for multiple tests, arguing that this association cannot be explained by population structure (Figure 6b). These results reveal the genetic signature of functional interaction (epistasis for fitness) between two physically unlinked genes in the same biochemical pathway, providing strong evidence that gch1 CNP results either directly or indirectly from antifolate selection.

Bottom Line: The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP.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.

View Article: PubMed Central - PubMed

Affiliation: Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX, USA.

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.

Show MeSH
Related in: MedlinePlus