Identification of ATP2B4 regulatory element containing functional genetic variants associated with severe malaria

Abstract

Genome-wide association studies (GWAS) for severe malaria have identified 30 genetic variants that are mostly located in non-coding regions, with only a few associations replicated in independent populations. In this study, we aimed at identifying potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium with the tagSNPs associated with severe malaria in several populations. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing 5 ATP2B4 SNPs in linkage disequilibrium with the tagSNP rs10900585. We confirmed the association of rs10900585 and also found significant associations of severe malaria with our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we showed that this region had both promoter and enhancer activities and that both individual SNPs and the combination of SNPs had regulatory effects using luciferase reporter assays. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Taken together, our data show that severe malaria-associated genetic variants alter the activity of a promoter with enhancer function. We showed that this regulatory element controls the expression of ATP2B4 that encodes a plasma membrane calcium-transporting ATPase 4 (PMCA4), which is the major calcium pump on red blood cells. Altering the activity of this regulatory element affects the risk of severe malaria probably through calcium concentration effect on parasitaemia.

Unraveling a novel dual-function regulatory element showing epistatic interaction with a variant that escapes genome-wide association studies

“…In this context, we have established a comprehensive pipeline that integrates genetic and epigenomic data using a combination of bioinformatics and experimental approaches. Our previous research resulted in the identification of an Epromoter within the ATP2B4 gene locus 17 , demonstrating the efficacy of our strategy. This Epromoter comprises five regulatory variants -namely rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255-all of which have been associated with severe malaria 17,21 and are in strong linkage disequilibrium (LD) with the nonfunctional lead SNP (rs10900585) previously identified by genome-wide association studies (GWAS) [22][23][24][25][26] .…”

“…Our previous research resulted in the identification of an Epromoter within the ATP2B4 gene locus 17 , demonstrating the efficacy of our strategy. This Epromoter comprises five regulatory variants -namely rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255-all of which have been associated with severe malaria 17,21 and are in strong linkage disequilibrium (LD) with the nonfunctional lead SNP (rs10900585) previously identified by genome-wide association studies (GWAS) [22][23][24][25][26] . The current challenge is to establish the link between this disease-associated region and the different genes affected, while also identifying the specific cell types involved.…”

“…Furthermore, such causal regulatory variants, which often reach a high frequency in populations, individually have a low impact on gene expression and are likely to affect disease risk through modest phenotypic effects. We, therefore, propose that functional assessment of common non-coding variants is often more relevant when several variants are tested simultaneously in order to assess their epistatic interaction and functional consequences 17,21 . Here, we found that the enhancer or silencer function is independent of the ATP2B4 haplotype in the presence of the major T allele at rs11240391 in Jurkat T cells, whereas the repressive function of the ESpromoter is stronger in combination with the minor G allele for rs11240391, suggesting an epistatic interaction between all these SNPs.…”

“…Considering the established involvement of the ESpromoter in LAX1 gene regulation and the physical interaction between it and the LAX1 promoter, we decided to explore potential epistatic interactions between rs11240391 and the five SNPs of the ESpromoter previously associated with severe malaria 17 . Individuals carrying both the GG risk genotype at rs11240391 and the ATP2B4 risk haplotype (Major haplotype) had a higher risk of developing severe malaria with 21% and 8% in severe malaria and controls, respectively (OR = 3.05, p < 0.0001) (Figure 6F, Figure S1, Table S2), suggesting an epistatic interaction between them.…”

“…These enhancers can activate gene transcription over large distances and independently of orientation. Recent studies have unveiled that a subset of promoters can also serve as enhancers, termed "Epromoters" [15][16][17][18] . Investigating these promoters, enhancers or Epromoters is essential for understanding complex gene regulation, as they wield the power to directly influence physiological traits or diseases by simultaneously regulating the expression of numerous proximal and distal genes.…”