Title: Role of ATP2B4 in human malaria: looking for functional genetic variants associated with malaria.
Date and place: September 11th 2020
Malaria causes approximately one million fatalities per year, mostly among Africa. The severity of the disease is influenced by complex interactions between many factors including host genetics. My thesis work concerns the identification and the functional characterization of the genetic variants involved in resistance/susceptibility to severe malaria (SM) by combining bioinformatics and genetic approaches. Several GWAS of SM have identified various loci. Nevertheless, only a limited number of loci and genetic variants have been replicated in independent populations. The first part of my project was to systematically identify the regulatory effect of all the SNPs in linkage disequilibrium (LD) with the tagSNPs associated with SM in several populations. By using different bioinformatics tools, we prioritized the potential causal variants and interestingly found 5 best candidates (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) located close to each other on chromosome 1 in ATP2B4 region. Our bioinformatic results predicted that they have a regulatory effect, whereas the tagSNPs are unlikely functional. ATP2B4 is the main calcium pump of erythrocytes (the host cells of the pathogenic stage of malaria parasites) and alteration of its expression may affect the intraerythrocytic calcium concentration and thus the structure and development of intraerythrocytic stages of the parasite. By genotyping these SNPs in a Senegalese population, we confirmed the association of tagSNP (rs10900585) and the 5 candidates (major allele as the risk allele) with SM. Moreover, a meta-analysis confirmed the association of this tagSNP with SM in several populations.
Furthermore, we have genotyped ATP2B4 variants (rs10751450, rs10751451, rs10752452) that have shown to be in an enhancer and to alter ATP2B4 expression and assessed their association with SM. Interestingly, we evidenced a close LD between these SNPs and rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255, and detected a significant association with SM in the Senegalese population. Moreover, haplotype analysis revealed that the individuals homozygous for the major haplotype were found to have a higher risk of developing SM. In the next part, we investigated the functional impact of the candidate variants on gene regulation by using different functional approaches. Luciferase assay results indicated a significant promoter activity for both risk (major alleles) and the non-risk haplotype (minor alleles) with non-risk haplotype inducing higher transcriptional activity. Furthermore, the region showed a significant enhancer effect on the ATP2B4 promoter.
However, the major haplotype (risk) exhibited significantly higher enhancer impact than minor haplotype (non-risk) which confirmed that the regulatory region exhibits allele-dependent enhancer activity in K562 and suggested that our regulatory region is an epromoter (promoter with enhancer function). Using CRISPR-Cas9, we performed the knockout of the region containing the SNPs and found a decrease in expression of the gene compared to WT for total expression as well as for two long transcripts, which confirmed that our regulatory region with rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255 has an enhancer function. Additionally, the impact of deletion on intracellular calcium concentration was measured using flow cytometry and the results indicated a significant increase in intracellular calcium in deleted clones. In conclusion, we identified an enhancer that controls ATP2B4 expression and is perturbed by several genetic variants associated with severe malaria. This also fosters the development of therapeutic strategies based on the modulation of ATP2B4 expression and calcium levels.
- Dr. Pascal Rihet (Thesis supervisor)
- Dr. Sandrine Marquet (Co-supervisor)
- Dr. Anavaj Sakuntabai (Reporter)
- Dr. Richard Redon (Reporter )
- Dr. Laurence Flori (Examiner)
- Dr. Catherine Nguyen (Examiner)