(P21-009-25) Exploring the Impact of a Single Genetic Variant on Cellular Zinc Homeostasis

Objectives: A genome-wide association study (GWAS) of 2,603 Australian twins and relatives revealed a significant correlation between rs2120019 in the PPCDC gene locus on chromosome 15 and erythrocyte zinc concentrations. Despite the prevalence of the minor ‘C’ allele—gnomAD: global (30%) and admixed American (27%); TOPMed: global (32%)—, the role of rs2120019 or PPCDC-associated metabolism in zinc homeostasis remains unexplored. This study aims to decipher the causation of rs2120019 in cellular zinc homeostasis using molecular genetics approaches.

Methods: Using a CRISPR-based Prime-Editing system, we introduced the minor ‘C’ allele of rs2120019 into the human leukemia HAP1 cell line, originally carrying the major ‘T’ allele. Stable HAP1 cell lines were isolated and validated for wild-type (WT; T/T), heterozygous (T/C), and homozygous (C/C) genotypes using Sanger sequencing. The expression of the predicted target genes of rs2120019, identified through expression quantitative trait loci (eQTL) data—PPCDC, MAN2C1, SCAMP2, SNUPN, FAM219B, RPP25, ULK3, and PBX1—were analyzed using RT-qPCR. PPCDC-overexpressing cells were generated by transfecting a PPCDC-cloning plasmid. Total cellular and free zinc levels were measured using ICP-OES and live cell imaging with fluorescent probe Zinpyr-1, respectively. Statistical analyses were performed using GraphPad Prism 10.

Results: The cell line homozygous for the minor allele C/C exhibited increased PPCDC gene expression and decreased expression of SCAMP2 and PBX1 compared to WT (T/T) and T/C cells. Total zinc levels in C/C cells were significantly reduced, showing a similar downtrend to the PPCDC-overexpressing cells when compared to WT or empty vector-transfected cells, respectively. Live cell fluorescent imaging revealed a significant reduction in the free zinc pool in C/C cells.

Conclusions: The observed reduction in total and free zinc levels in cells carrying the rs2120019 minor allele compared to the major allele supports the SNP’s causal impact on zinc homeostasis and aligns with previous GWAS findings on its association with erythrocyte zinc concentrations. Future research will elucidate how rs2120019 regulates cellular and systemic zinc homeostasis.

Funding Sources: Partially supported by Food and Human Health (USDA NIFA) and the Department of Nutritional Sciences, University of Connecticut.