A Single Genetic Locus Controls Both Expression of DPEP1/CHMP1A and Kidney Disease Development Via Ferroptosis

ARTICLE https://doi.org/10.1038/s41467-021-25377-x OPEN A single genetic locus controls both expression of DPEP1/CHMP1A and kidney disease development via ferroptosis Yuting Guan 1,2,9, Xiujie Liang1,2,9, Ziyuan Ma 1,2, Hailong Hu1,2, Hongbo Liu 1,2, Zhen Miao 1,2,3, ✉ Andreas Linkermann 4,5, Jacklyn N. Hellwege 6, Benjamin F. Voight 2,7,8 & Katalin Susztak 1,2 fi 1234567890():,; Genome-wide association studies (GWAS) have identi ed loci for kidney disease, but the causal variants, genes, and pathways remain unknown. Here we identify two kidney disease genes Dipeptidase 1 (DPEP1) and Charged Multivesicular Body Protein 1 A (CHMP1A) via the triangulation of kidney function GWAS, human kidney expression, and methylation quantitative trait loci. Using single-cell chromatin accessibility and genome editing, we fine map the region that controls the expression of both genes. Mouse genetic models demonstrate the causal roles of both genes in kidney disease. Cellular studies indicate that both Dpep1 and Chmp1a are important regulators of a single pathway, ferroptosis and lead to kidney disease development via altering cellular iron trafficking. 1 Department of Medicine, Renal Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 2 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 3 Graduate group in Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 4 Division of Nephrology, Department of Internal Medicine, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Germany. 5 Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany. 6 Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Nashville, TN 37232, USA. 7 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 8 Institute of Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. ✉ 9These authors contributed equally: Yuting Guan, Xiujie Liang. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:5078 | https://doi.org/10.1038/s41467-021-25377-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-25377-x hronic kidney disease (CKD) affects 800 million people region and the expression of DPEP1 and CHMP1A (Fig. 1a, b). Cworldwide and remains the tenth leading cause of death. This eQTL effect was detected both in the tubule (n = 121) and Key molecular pathways that govern CKD pathogenesis glomerular (n = 119) compartments. remain largely unknown. Disease susceptibility shows substantial As both mQTL and eQTLs appeared to be tagged by rs164748, heterogeneity1, which is thought to be explained by environ- we next performed statistical colocalization to quantify the extent mental and genetic risk factors that have yet to be fully elucidated. to which causal genetic variants for kidney function, methylation, Genome-wide association analyses (GWAS) performed in large and gene expression were shared. Moloc analysis was performed populations identified nearly 250 loci where genetic variants with a+/− 100 kb window, covering most cis-regulatory inter- associated with kidney function2–4. Despite the success of locus actions. Molocalization analyses suggested a high posterior discovery, the causal variants, target genes, and cell types along probability that the eGFR GWAS, mQTL, and eQTL could be with the underlying molecular mechanisms remain unresolved, explained by common variants (PP_abc = 0.92, “Methods” mostly because more than 90% of GWAS identified variants are section). The moloc identified CpGs (cg21038338, cg11907426, non-coding5 and the variants are linked due to strong linkage cg07716468, cg01510696, and cg00487989) were kidney-specific disequilibrium6. mQTLs (when compared to whole blood15 and skeletal muscle)16 Prior GWAS functional annotation studies have highlighted the (Supplementary Data 1). By examining the association between enrichment of the causal variants in cell type-specificcis-regulatory GWAS variants and gene expression in a collection of micro- regions7,8. Epigenetic annotation has helped to narrow variants with dissected human kidney tubule and glomerulus samples, we the highest probability of regulatory function9.Onewayinwhicha found that the genotype of the top GWAS eGFR SNP (rs164748) GWAS variant could influence the nearby gene is by altering the strongly influenced the level of DPEP1 both in glomeruli and sequence where transcription factors (TFs) bind10. With this simple tubuli. Specifically, the GWAS risk allele G was associated with model in mind, contemporary approaches to identify a potential increased DPEP1 expression. Moreover, we found that the target gene have relied on the expression of quantitative trait loci genotype of this top GWAS eGFR SNP (rs164748) strongly analysis (eQTL), where genetic variants and gene expression influenced the expression level of CHMP1A in tubuli. The GWAS changes are correlated in a tissue-specific manner11. risk allele G was associated with lower CHMP1A expression Due to the strong linkage disequilibrium multiple variants at a (Fig. 1c). We noted that the tubule and glomerular eQTL effects single locus show strong association with disease development. at rs164748 on DPEP1 and the glomerular eQTL effect on Initial locus dissection studies focused on defining a single causal CHMP1A replicated in the publicly available NephQTL variant responsible for disease development. New experimental database17, which contained only 136 samples (Fig. S1b). In the locus dissection studies indicate that it is possible that multiple GWAS, the C allele of rs164748 was associated with high eGFR, SNPs at a single locus is responsible for gene regulation. Fur- while in the eQTL, the C allele was associated with lower DPEP1 thermore, it is also tempting to speculate that multiple GWAS and higher CHMP1A expression. causal variants regionally implicate the same causal gene, this We next examined the potential eQTL effects of rs164748 in may not be true in all cases. In addition, genes are not randomly the GTEx project18. While we did note an association between the distributed in the genome. Genes with similar functions are often CKD risk genotype and CHMP1A expression in skin samples clustered together12, such genic ‘operons’ may share regulatory (Supplementary Data 2), we did not observe an association regions in common, raising the possibility that multiple genes between genetic variation of rs164748 and expression of DPEP1 within an individual locus contribute causally to the underlying in any GTEx tissue (Supplementary Data 2). We also did not phenotype. The extent to which multiple phenotype-causal genes observe an association between rs164748 genotype and expres- are driven by a single or multiple causal variant at a single locus is sion of CDK10 and SPATA33 in kidney samples (Fig. S1b–d). not fully known. Collectively, these results suggest a kidney-specific mQTL/eQTL In this study, we computationally integrated kidney function effect of rs164748, which prioritized both DPEP1 and CHMP1A GWAS, methylation, expression quantitative trait loci, and as kidney disease risk genes. human kidney single nuclei ATAC sequencing studies. Our analysis prioritized both DPEP1 and CHMP1A as kidney disease risk genes in kidney proximal tubules, likely mediating the effect Human kidney-specific single nuclei chromatin accessibility of the eGFR GWAS signal observed on chromosome 16. Using analysis highlights likely causal variants in kidney proximal CRISPR mediated genome deletion, we showed that both genes tubules. To narrow likely causal variants, we utilized human were regulated by a common genomic locus in human kidney kidney single nuclei sequencing assay for transposase-accessible proximal tubule cells. Mouse gene knockouts highlighted the key chromatin (snATAC-seq). Interrogation of the snATAC-Seq data role of both genes in kidney disease development. Molecular at this locus identified 12 accessible chromatin regions. One studies indicated that both genes are regulators of ferroptosis. accessible chromatin region, nearby the 5′ end of CHMP1A, was present in all analyzed cells, likely corresponding to the promoter region19. The remaining 11 regions were only present in proximal Results tubule cells of the human kidney (Fig. 2a). Mouse kidney single- Shared causal variants for kidney function and CHMP1A/ cell accessible chromatin analysis showed important conservation DPEP1 expression. Previous GWAS have demonstrated robust of the locus (Fig. S2a), including the genomic organization, the and reproducible association of the chromosome 16 region tagged open chromatin at the promoter region, and the proximal tubule- by rs1647483,13,14 with kidney function (eGFR) (Fig. 1a and specific open chromatin pattern. Human kidney bulk H3K27ac, Fig. S1a). This locus spans multiple genes, which include DPEP1, H3K4me1, and H3K4me3 chromatin immunopre-cipitation CHMP1A, SPATA33, and CDK10. To dissect this region, we (ChIP-seq) information further supported the single-cell data integrated the GWAS data with kidney methylation quantitative (Fig. 2a). By direct overlapping the kidney disease-associated trait loci and (mQTL) and eQTL, respectively. First, we observed

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