Fig 4. Pfcyp19b promoter region harbors short tandem repeat polymorphism that affects PfCYP19B expression levels.

(A) Graphical representation of 1.5Kb-long intergenic region between falcipain 2a (FP2A) and pfcyp19b locus used in a dual luciferase reporter assay. Cyclophilin’s start codon is shown in red and the predicted 5’UTR in green. 426 polymorphic STR site has been highlighted in blue and has been magnified to show all distinct alleles detected in TRACI and TRACII genome analyses. The mountain chart (above) illustrates GC content (in % of GC) across the region. 426 STR is in the middle of AT-rich area just before the start of 5’UTR. (B-D) Pfcyp19b expression values according to detected 426 STR allele variants (dot plots, left panels) from TRACI WGS (n = 462—subfigure B) and TRACII (n = 87 for WGS—subfigure C; n = 144 for amplicon sequencing—subfigure D). Average parasite PC1/2 values for each STR variant are indicated above the dot plots. Charts on the right show geographic distribution (in %) of each STR variant in each GMS country. In TRACI ^del(7xAT) variant was predominantly present in highly artemisinin-resistant Cambodian samples whereas ^ref3D7 was a dominant allele in the whole GMS region. In TRACII ^del(7xAT) variant spread across entire GMS region including still artemisinin-sensitive wGMS (Bangladesh, Myanmar). All p-values are derived from unpaired t-test with Welch’s correction performed individually between ^ref3D7 and respective STR variants (E) Dual luciferase reporter assay of pfcyp19b promoter with genetically engineered STRs at position 426bp upstream of pfcyp19b start codon. Firefly luciferase signal has been normalized to Renilla luciferase used as a control for transfection and expression efficiency. All p-values are derived from unpaired t-test with Welch’s correction between ^ref3D7 and respective STR variants where: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. All transient transfection dual luciferase experiments were performed in six independent biological replicates.