Namrata Madan, Andrew R. Ghazi, Xianguo Kong, Edward S. Chen, Chad A. Shaw et al.
CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies.
Namrata Madan, Andrew R. Ghazi, Xianguo Kong, Edward S. Chen, Chad A. Shaw, Leonard C. Edelstein. Functionalization of CD36 cardiovascular disease and expression associated variants by interdisciplinary high throughput analysis. 2019-07-25. https://doi.org/10.1371/journal.pgen.1008287
An episomal plasmid MPRA assayed 81 platelet CD36 eQTL SNPs and 10 known regulatory positive-control constructs in human K562 cells, with 40 unique 10-bp barcodes per allele or construct. The library was transfected and RNA harvested 48 hours later; this table reports the published allele/construct transcription shifts and t-test, Mann–Whitney U-test, and Bayesian model statistics.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 9 files (ZIP)supplement_S1_complete_mpra_positive_controls.pdfsupplement_S1_figure_activity_scatterplots.pdfsupplement_S2_complete_mpra_cd36_variants.pdfsupplement_S2_figure_hypothesized_mechanism.pdfsupplement_S3_figure_barcode_representation.pdfsupplement_S3_mpra_control_sequences.pdfsupplement_S4_figure_bayesian_model.pdfsupplement_S4_rnaseq_quality.pdfsupplement_S5_luciferase_sequences.pdf