An Essential Erythroid-Specific Enhancer of ATP2B4 Associated with Red Blood Cell Traits and Malaria Susceptibility

Genome-wide association studies (GWASs) have identified thousands of common genetic variants associated with human traits and disease susceptibility. Given that the majority of GWAS-identified SNPs are located within non-coding regions of the genome, the mechanisms of these trait associations are frequently unknown. Here we investigate the biological underpinnings of common trait-associated genetic variation at ATP2B4, a gene on chromosome 1q32 that encodes a major plasma membrane calcium ATPase (also known as PMCA4). Genetic variation within this locus is associated with several phenotypes including various red blood cell traits (mean corpuscular hemoglobin concentration and red cell distribution width) as well as susceptibility to severe malaria infection. We conducted an expression quantitative trait loci (eQTL) mapping analysis in human erythroblasts and identified a set of SNPs associated with ATP2B4 gene expression in cis. These included the same genetic variants found by GWAS to be associated with RBC traits and malaria susceptibility. Furthermore, these SNPs overlap an intronic erythroid DNase I hypersensitive site at ATP2B4. An analysis of the ENCODE database showed that this element was erythroid specific in that it lacked DNase I hypersensitivity in >30 queried non-erythroid cell types. We used the CRISPR/Cas9 genome editing system to delete this element in HUDEP-2 immortalized human erythroid precursor cells. We observed that cells bearing a 927 bp biallelic deletion of this noncoding element displayed near complete loss of ATP2B4 expression (3% residual gene expression), while cells bearing heterozygous deletions showed an intermediate gene expression phenotype. We identified a core element that encompassed 3 of the highly trait associated SNPs and 5 GATA1-binding motifs. Biallelic deletion of this 98 bp core led to 83% reduction in ATP2B4 expression. Disruption of individual GATA1-binding motifs resulted in partial reduction of gene expression, suggesting the contribution of multiple binding sites to appropriate gene expression. Overall, this study suggests that variation within an essential erythroid-specific enhancer of ATP2B4 underlies the association of ATP2B4 with RBC traits and malaria susceptibility. Furthermore these results encourage combined analyses of gene expression, chromatin state, and prospective genetic perturbation as a means to explore the variants, elements and genes responsible for heritable blood phenotypes.