Genomic discovery and functional validation of MRP1 as a novel fetal hemoglobin modulator and potential therapeutic target in sickle cell disease

Sickle cell disease (SCD) remains a major health burden with limited treatment options. Despite promising gene-editing clinical trials, there is an unmet need for cost-effective therapies. As induction of fetal hemoglobin (HbF) is an established therapeutic strategy for SCD, we conducted a genome-wide association study of circulating HbF levels in ~11,000 participants to identify further HbF modulators. We identified associations in 11 genomic regions, including eight novel loci such as ABCC1 (encoding multidrug resistance-associated protein 1, MRP1). Using gene-editing and pharmacological approaches, we showed that inhibition of MRP1 increases HbF, intracellular glutathione levels, and reduces sickling in erythroid cells from SCD patients. Overall, our findings identify several novel genetically-validated potential therapeutic targets for SCD, including promising proof-of-principle results from small molecule inhibition of MRP1. ### Competing Interest Statement John Danesh serves on scientific advisory boards for AstraZeneca, Novartis, and UK Biobank, and has received multiple grants from academic, charitable and industry sources outside of the submitted work. Adam Butterworth reports institutional grants from AstraZeneca, Bayer, Biogen, BioMarin, Novartis, Regeneron, Sanofi and Bioverativ (a Sanofi company). During the drafting of the manuscript, Dirk Paul became a full-time employee of AstraZeneca and Lisa Schmunk became a full-time employee of Chronomics Ltd. Yannis Hara, Samuel Lessard and Alexandra Hicks are employees and stakeholders of Sanofi. Sriram Krishnamoorthy is an employee of Cellarity. ### Funding Statement The fetal hemoglobin measurements in INTERVAL were funded by Biogen. Experimental follow-up was supported by a grant from the Sanofi iAwards Europe Program. Participants in the INTERVAL randomised controlled trial were recruited with the active collaboration of NHS Blood and Transplant England ([www.nhsbt.nhs.uk][1]), which has supported field work and other elements of the trial. DNA extraction and genotyping were co-funded by the National Institute for Health and Care Research (NIHR), the NIHR BioResource () and the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014) [*]. The academic coordinating centre for INTERVAL was supported by core funding from the: NIHR Blood and Transplant Research Unit in Donor Health and Genomics (NIHR BTRU-2014-10024), NIHR BTRU in Donor Health and Behaviour (NIHR203337), UK Medical Research Council (MR/L003120/1), British Heart Foundation (SP/09/002; RG/13/13/30194; RG/18/13/33946) and NIHR Cambridge BRC (BRC-1215-20014) [*]. A complete list of the investigators and contributors to the INTERVAL trial is provided in reference [**]. The academic coordinating centre would like to thank blood donor centre staff and blood donors for participating in the INTERVAL trial. Professor John Danesh holds a British Heart Foundation Professorship and a NIHR Senior Investigator Award [*]. This work was supported by Health Data Research UK, which is funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation and Wellcome. *The views expressed are those of the author(s) and not necessarily those of the NIHR, NHSBT or the Department of Health and Social Care. **Di Angelantonio E, Thompson SG, Kaptoge SK, Moore C, Walker M, Armitage J, Ouwehand WH, Roberts DJ, Danesh J, INTERVAL Trial Group. Efficiency and safety of varying the frequency of whole blood donation (INTERVAL): a randomised trial of 45 000 donors. Lancet. 2017 Nov 25;390(10110):2360-2371. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Research Ethics Service Committee East of England - Cambridge East (Research Ethics Committee (REC) reference 11/EE/0538) - gave ethical approval for the INTERVAL study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Upon publication, the INTERVAL HbF GWAS summary statistics will be made publicly available in the NHGRI-EBI GWAS Catalog () and the RNA sequencing data will be available via the Gene Expression Omnibus portal (). [1]: http://www.nhsbt.nhs.uk