Genetic and epigenetic changes in host ABCB1 influences malaria susceptibility to Plasmodium falciparum

Abstract

Multiple mechanisms such as genetic and epigenetic variations within a key gene may play a role in malarial susceptibility and response to anti-malarial drugs in the population. ABCB1 is one of the well-studied membrane transporter genes that code for the P-glycoprotein (an efflux protein) and whose effect on malaria disease predisposition and susceptibility to drugs remains to be understood. We studied the association of single nucleotide variations in human ABCB1 that influences its function in subjects with uncomplicated and complicated malaria caused by Plasmodium falciparum (Pf). Global DNA methylation and ABCB1 DNA promoter methylation levels were performed along with transcriptional response and protein expression in subjects with malaria and healthy controls. The rs2032582 locus was significantly associated with complicated and combined malaria groups when compared to controls (p < 0.05). Significant DNA methylation difference was noticed between case and control (p < 0.05). In addition, global DNA methylation levels of the host DNA were inversely proportional to parasitemia in individuals with Pf infection. Our study also revealed the correlation between ABCB1 DNA promoter methylation with rs1128503 and rs2032582 polymorphisms in malaria and was related to increased expression of ABCB1 protein levels in complicated malaria group (p < 0.05) when compared to uncomplicated malaria and control groups. The study provides evidence for multiple mechanisms that may regulate the role of host ABCB1 function to mediate aetiology of malaria susceptibility, prognosis and drug response. These may have clinical implications and therapeutic application for various malarial conditions.

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.

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).

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.