Table 1.
Demographic data and the clinical profile of the malaria patients recruited as participants for the study.
Fig 1.
Schematic representation showing genome organization of ABCB1 gene and the analyzed regions for SNPs and CpG sites (indicated in blue).
Exons are indicated as boxes.
Fig 2.
Western blotting analysis of anti-ABCB1 antibody and β-actin was used as internal control.
(Lane 1: Individual with complicated malaria; Lane 2: Individual with un-complicated malaria; Lane 3: Healthy individual).
Fig 3.
Figure shows PCR-RFLP (upper half) and confirmatory DNA sequencing (lower half) experiments, for the three SNPs [rs1128503 (panel A), rs2032582 (panel B) and rs1045642 (panel C)] of ABCB1 gene. Panel A shows agarose gel electrophoresis of PCR amplicons (size, 366 bp) containing rs1128503 C>T polymorphism, digested with restriction enzyme HaeIII showing homozygous wild type CC individuals (lane 2) homozygous mutant type TT alleles (lane 3) and heterozygous CT variant (lane 4). The undigested PCR amplicon is shown in lane 1 and the DNA size marker is shown in lane 5. Panel B shows PCR-RFLP with BanI restriction enzyme for genotyping rs2032582G>T polymorphism. The wild type allele G containing PCR amplicon (of size, 224 bp) is digested by BanI to generate two fragments of 198 and 26 base pairs. The three genotypes corresponding to the fragment lengths, GG (lane 6), GT (lane 2) and TT (lane 3, 4, 5 and 7) are shown in the picture. Panel C shows agarose gel electrophoresis of PCR amplicons (size 197 bp) containing rs1045642 C>T polymorphism, digested with restriction enzyme Sau3A1 showing homozygous wild type CC individuals (lane 5) homozygous mutant type TT alleles (lane 2, 3) and heterozygous CT variant (lane 4). Here, the PCR amplicons with wild type C allele is digested into two fragments of size 158 bp and 39 bp whereas the mutant T allele containing fragment remain undigested. DNA sequencing results for each polymorphism are presented in the bottom half of the panels in the figure as the DNA sequences of wild type homozygotes, heterozygotes and mutant homozygotes of the three polymorphisms represented in the respective panels as indicated by arrows.
Table 2.
Genotype/allele frequency data of SNPs of ABCB1 gene and the results of the test for genetic association with case (uncomplicated, complicated and all malaria) and control, with measures of statistical significance.
Table 3.
Predicted haplotypes of the SNPs of ABCB1 gene, and statistical analysis of the association to complicated, uncomplicated and malaria patients versus controls.
Fig 4.
Dot plot of ABCB1 promoter DNA methylation at 30 CpG sites.
Complicated malaria (Panel A), Uncomplicated malaria (Panel B), All malaria cases (Panel C) and Controls (Panel D).
Table 4.
Results obtained from One-way ANOVA test of DNA methylation levels of ABCB1 promoter in complicated, uncomplicated, all malaria and control groups.
Table 5.
Correlation analysis of the three SNPs of ABCB1 gene and DNA methylation levels in all malaria cases and controls.
Fig 5.
Global methylation estimation by RP-HPLC.
A. Global methylation values (5mC) in malaria patients (with and without complications) and controls. Each dot represents the total methylation cytosine content of the individual sample. B. Global methylation values (5mC) in malaria patients (with different parasite levels). C. Global methylation values (5mC) in malaria patients (before and after treatment). Patient blood was collected twice at the time of admission (before medication) and after 7days of treatment. Within the groups mean ± standard error was shown in lines. The aster sign indicates the p value significance. **P<0.01, *P<0.05.
Fig 6.
Promoter activity analysis of ABCB1 constructs using luciferase assays.
Showing pGL3- ABCB1 construct (contains 949bp DNA fragment) promoter activity results upon artemisinin and 3-methylcholanthrene treatments.