Abstract
Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.
| Original language | English |
|---|---|
| Pages (from-to) | 135-146 |
| Number of pages | 12 |
| Journal | Blood |
| Volume | 141 |
| Issue number | 2 |
| Early online date | 21 Sept 2022 |
| DOIs | |
| Publication status | Published - 23 Jan 2023 |
Bibliographical note
Funding Information:This study was supported in part by the National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Cell Products (IS-BTU-1214-10032), NHSBT R&D funding (WP15-04 and WP-15-05), the Medical Research Council (MR/V010506/1), the Cultural Bureau at the Royal Saudi Embassy in the United Kingdom and the Saudi Ministry of Education (SAA PhD scholarship), and the European Union ITN “EVIDENCE” grant agreement ID 860436)
Funding Information:
The authors thank the following individuals: Jan Frayne, Dave Anstee, and Kongtana Trakarnsanga for provision of the BEL-A cell line; Kate Heesom from the University of Bristol Proteomics Facility for mass spectrometry analysis of immune precipitates; Andrew Herman from the University of Bristol Faculty of Biomedical Sciences Flow Cytometry Facility for cell sorting support; IBGRL Molecular Diagnostics Department for Sanger sequencing support; Christian Jannete Stevens, from Bristol Institute of Transfusion Sciences, UK, for providing PIEZO1 amplification primers for exons 23, 24, and 39; and Geoff Daniels for critical review of the manuscript. The authors would also like to acknowledge the invaluable insights, advice, and support from Dave Anstee (1946-2021). This study was supported in part by the National Institute for Health Research Blood and Transplant Research Unit (NIHR BTRU) in Red Cell Products (IS-BTU-1214-10032), NHSBT R&D funding (WP15-04 and WP-15-05), the Medical Research Council (MR/V010506/1), the Cultural Bureau at the Royal Saudi Embassy in the United Kingdom and the Saudi Ministry of Education (SAA PhD scholarship), and the European Union ITN “EVIDENCE” grant agreement ID 860436), The views expressed are those of the authors and not necessarily those of the National Health Service, NIHR, or Department of Health and Social Care. Contribution: N.M.T. V.K.C. L.A.T. T.J.S. and A.M.T. conceived, designed, and coordinated the study; N.M.T. and B.J. performed serology experiments; L.A.T. V.K.C. and S.A.A. performed whole exome sequencing, Sanger sequencing, and associated data analysis; T.J.S. designed and performed CRISPR/Cas9 experiments (knockout and overexpression of Piezo1) and performed the flow cytometry experiments; P.J.W. carried out protein modeling and molecular dynamics calculations; F.A.S. and S.A.A. designed and carried out immunoprecipitation experiments; C.M.F. N.M. and M.G.R. conducted patch clamping optimization, experiments, and analysis under the supervision of N.B. and M.R.; S.J.W. detected and provided P11, P11-F1, and P11-F2 samples for antibody identification; M.H. R.A. and M.A. detected and referred samples P12 and P12-F1 to P12-F11 for antibody identification and provided clinical information; I.S. S.L. S.H. and J.J. detected and referred samples P13, P13-F1 and P13-F2 for antibody investigation; T.L. W.L. and M.D.K. provided comprehensive clinical data and interpretation for the case of P13; A.M.T. and N.M.T. provided advice on experimental design, analysis and interpretation of data, manuscript preparation, and critical review; N.M.T. V.K.C. L.A.T. S.A.A. and T.J.S. wrote the manuscript; and all authors contributed to review of the final manuscript.
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