Identification and single-base gene-editing functional validation of a cis-EPO variant as a genetic predictor for EPO-increasing therapies
Harlow, Charli E; Gandawijaya, Josan; Bamford, Rosemary A; Martin, Emily-Rose; Wood, Andrew R; van der Most, Peter J; Tanaka, Toshiko; Leonard, Hampton L; Etheridge, Amy S; Innocenti, Federico; Beaumont, Robin N; Tyrrell, Jessica; Nalls, Mike A; Simonsick, Eleanor M; Garimella, Pranav S; Shiroma, Eric J; Verweij, Niek; van der Meer, Peter; Gansevoort, Ron T; Snieder, Harold; Gallins, Paul J; Jima, Dereje D; Wright, Fred; Zhou, Yi-Hui; Ferrucci, Luigi; Bandinelli, Stefania; Hernandez, Dena G; van der Harst, Pim; Patel, Vickas V; Waterworth, Dawn M; Chu, Audrey Y; Oguro-Ando, Asami; Frayling, Timothy M
(2022) American Journal of Human Genetics, volume 109, issue 9, pp. 1638 - 1652
(Article)
Abstract
Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are currently under clinical development for treating anemia in chronic kidney disease (CKD), but it is important to monitor their cardiovascular safety. Genetic variants can be used as predictors to help inform the potential risk of adverse effects associated with drug treatments. We therefore
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aimed to use human genetics to help assess the risk of adverse cardiovascular events associated with therapeutically altered EPO levels to help inform clinical trials studying the safety of HIF-PHIs. By performing a genome-wide association meta-analysis of EPO (n = 6,127), we identified a cis-EPO variant (rs1617640) lying in the EPO promoter region. We validated this variant as most likely causal in controlling EPO levels by using genetic and functional approaches, including single-base gene editing. Using this variant as a partial predictor for therapeutic modulation of EPO and large genome-wide association data in Mendelian randomization tests, we found no evidence (at p < 0.05) that genetically predicted long-term rises in endogenous EPO, equivalent to a 2.2-unit increase, increased risk of coronary artery disease (CAD, OR [95% CI] = 1.01 [0.93, 1.07]), myocardial infarction (MI, OR [95% CI] = 0.99 [0.87, 1.15]), or stroke (OR [95% CI] = 0.97 [0.87, 1.07]). We could exclude increased odds of 1.15 for cardiovascular disease for a 2.2-unit EPO increase. A combination of genetic and functional studies provides a powerful approach to investigate the potential therapeutic profile of EPO-increasing therapies for treating anemia in CKD.
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Keywords: CRISPR-Cas9, anaemia in chronic kidney disease, cardiovascular disease risk, drug-target mendelian randomization, functional genomics, functional validation, gene editing, genome-wide association study, mendelian randomization, population studies, statistical genetics, Genetics(clinical), Genetics
ISSN: 0002-9297
Publisher: Cell Press
Note: Funding Information: The authors acknowledge all participants and investigators involved in the different studies. The HealthABC study was supported by National Institute on Aging (NIA) contracts (N01-AG-6-2101; N01-AG-6-2103; N01-AG-6-2106; NIA grant R01-AG0280502, and NINR grant R01-NR012459) and was funded in part by the Intramural Research Program of the NIA. The InCHIANTI study baseline (1998–2000) was supported as a “targeted project” (ICS110.1/RF97.71) by the Italian Ministry of Health and in part by the NIA (contracts: 263 MD 9164 and 263 MD 821336). The research utilized data from the UK Biobank (UKB) resource carried out under application number 9072. UKB protocols were approved by the National Research Ethics Service Committee. The authors thank the Exeter Sequencing service in carrying out the RNA-sequencing. The equipment utilized is funded by the Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Award (WT101650MA), and BBSRC LOLA award (BB/K003240/1). The authors acknowledge the use of the University of Exeter High-Performance Computing (HPC) facility in carrying out this work funded by an MRC Clinical Research Infrastructure award (MRC grant: MR/M008924/1). T.M.F. is supported by MRC awards MR/WO14548/1 and MR/T002239/1. J.T is funded by an Academy of Medical Sciences grant. pLV-EF1alpha-EPO-218-PDGFR was a gift from Tao Liu. The authors thank Akshay Bhinge for providing the piggyBac plasmid and transposase plasmid, Dario Pacitti for his help with the RNA-seq analysis, and Natsuki Takamura and Yuriko Iizuka for help in the laboratory. For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. C.E.H. was awarded an MRC iCASE studentship (MR/P016065/1), which was cofunded by GSK and the MRC for the duration of the study. GSK are undertaking clinical development into a novel PHI and have given permission to publish this work alongside an internal review of the manuscript. A.Y.C. is an employee of GSK, is a shareholder of GSK stock, and was involved in the study design, interpretation of the data, and writing of the paper. V.V.P. was an employee of GSK at the time of the study conception and design. He was involved in interpreting the data and writing of the paper and is a shareholder of GSK and Roche Holding AG stocks. D.M.W. was an employee of GSK at the time of the study conception and design and was involved in the interpretation of the data and writing of the paper. H.L.L. and M.A.N. received support from a consulting contract between Data Tecnica International and the National Institute on Aging (NIA), National Institutes of Health (NIH). They were involved in data analysis and review of the manuscript. N.V. is a full-time employee of Regeneron Pharmaceutical Inc. and receives stock options and restricted stock units as compensation. D.D.J. received support from the Center of Human Health and the Environment P30ESO25128 grant. F.I. is an AbbVie employee and receives stocks from AbbVie, which has not been involved in this work at any level. Funding Information: The authors acknowledge all participants and investigators involved in the different studies. The HealthABC study was supported by National Institute on Aging (NIA) contracts ( N01-AG-6-2101 ; N01-AG-6-2103 ; N01-AG-6-2106 ; NIA grant R01-AG028050 2, and NINR grant R01-NR012459 ) and was funded in part by the Intramural Research Program of the NIA. The InCHIANTI study baseline (1998–2000) was supported as a “targeted project” ( ICS110.1/RF97.71 ) by the Italian Ministry of Health and in part by the NIA (contracts: 263 MD 9164 and 263 MD 821336 ). The research utilized data from the UK Biobank (UKB) resource carried out under application number 9072. UKB protocols were approved by the National Research Ethics Service Committee . The authors thank the Exeter Sequencing service in carrying out the RNA-sequencing. The equipment utilized is funded by the Wellcome Trust Institutional Strategic Support Fund ( WT097835MF ), Wellcome Trust Multi User Equipment Award ( WT101650MA ), and BBSRC LOLA award ( BB/K003240/1 ). The authors acknowledge the use of the University of Exeter High-Performance Computing (HPC) facility in carrying out this work funded by an MRC Clinical Research Infrastructure award (MRC grant: MR/M008924/1 ). T.M.F. is supported by MRC awards MR/WO14548/1 and MR/T002239/1 . J.T is funded by an Academy of Medical Sciences grant. pLV-EF1alpha-EPO-218-PDGFR was a gift from Tao Liu. The authors thank Akshay Bhinge for providing the piggyBac plasmid and transposase plasmid, Dario Pacitti for his help with the RNA-seq analysis, and Natsuki Takamura and Yuriko Iizuka for help in the laboratory. For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Funding Information: C.E.H. was awarded an MRC iCASE studentship (MR/P016065/1), which was cofunded by GSK and the MRC for the duration of the study. GSK are undertaking clinical development into a novel PHI and have given permission to publish this work alongside an internal review of the manuscript. A.Y.C. is an employee of GSK, is a shareholder of GSK stock, and was involved in the study design, interpretation of the data, and writing of the paper. V.V.P. was an employee of GSK at the time of the study conception and design. He was involved in interpreting the data and writing of the paper and is a shareholder of GSK and Roche Holding AG stocks. D.M.W. was an employee of GSK at the time of the study conception and design and was involved in the interpretation of the data and writing of the paper. H.L.L. and M.A.N. received support from a consulting contract between Data Tecnica International and the National Institute on Aging (NIA), National Institutes of Health (NIH). They were involved in data analysis and review of the manuscript. N.V. is a full-time employee of Regeneron Pharmaceutical Inc. and receives stock options and restricted stock units as compensation. D.D.J. received support from the Center of Human Health and the Environment P30ESO25128 grant. F.I. is an AbbVie employee and receives stocks from AbbVie, which has not been involved in this work at any level. Publisher Copyright: © 2022 The Author(s)
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