Identification and characterisation of SMIM1 variants determining the Vel blood group Christophersen, Mikael Kronborg 2017 Document Version: Publisher's PDF, also known as Version of record Link to publication Citation for published version (APA): Christophersen, M. K. (2017). Identification and characterisation of SMIM1 variants determining the Vel blood group. Lund University: Faculty of Medicine. 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LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Download date: 04. Oct. 2021 Identification and characterisation of SMIM1 variants determining the Vel blood group Mikael Kronborg Christophersen DOCTORAL DISSERTATION by due permission of the Faculty of Medicine, Lund University, Sweden. To be defended at 13:00 on Thursday 30th March 2017 at Biomedicinsk Centrum, Segerfalksalen Faculty opponent Associate Professor Morten Hanefeld Dziegiel Organization Document name LUND UNIVERSITY DOCTORAL DISSERTATION Department of Laboratory Medicine Date of issue Division of Hematology and Transfusion Medicine March 30th 2017 Author(s) Mikael Kronborg Christophersen Sponsoring organization Title and subtitle Identification and characterisation of SMIM1 variants determining the Vel blood group Abstract The Vel blood group antigen is present on red blood cells from all humans except rare Vel-negative individuals, who can form antibodies to Vel in response to transfusion or pregnancy. It was first described in 1952 as a high incidence antigen, while the molecular background was recently discovered to be a 17-bp deletion in Small Integral Membrane Protein 1, that causes a frame-shift mutation and abolishes SMIM1 expression, thus creating a Vel-negative phenotype. This thesis contains one of the three original discovery studies reporting the Vel antigen-defining SMIM1- deletion. We analysed SNP microarray data from Vel-positive and -negative individuals and identified SMIM1 as a potential gene. We then found the 17-bp deletion to only be present in Vel-negative individuals and sought to finally prove SMIM1 as the genetic background for the Vel antigen by examining: 1) SMIM1 mRNA sequence and expression levels, 2) presence of SMIM1 and the Vel antigen in red blood cell membranes from Vel-positive and -negative individuals and 3) anti-Vel antibody reactivity in erythroleukaemia cells expressing wild type and mutant SMIM1 protein. Our discovery allowed Vel to be officially recognised by the International Society of Blood Transfusion as blood group system 034. The SMIM1 deletion is the major determinant for Vel expression, yet even Vel-positive individuals (i.e. people carrying wild type SMIM1) show substantial variation in reactivity with anti-Vel antibody, creating a risk for Vel blood typing errors and transfusion reactions. We suspected the cause to be sequence variants in SMIM1 and found rs143702418 (insertion, C>CGCA) and rs1175550 (A>G) to independently influence expression of SMIM1, potentially mediated by the erythroid transcription factor TAL1 that binds preferentially to the high- expressing rs1175550G allele. Lastly, we examined historical Vel-negative samples and sought to retroactively reconcile historic Vel designations to our current knowledge of the SMIM1 deletion variant. As such, we found the old Vel;–1,–2 designation, attributed to individuals phenotyped to have weak to no Vel antigen expression, to correspond to homozygosity for the SMIM1 deletion, while persons designated Vel;1,–2, low Vel antigen expression, matched with being heterozygous carrier of the deletion. The Vel antigen was one of the few remaining clinically significant antigens with unknown genetic background, that causes severe haemolytic transfusion reactions. This thesis assisted in characterising the molecular background of the Vel antigen, which paved the way for molecular Vel typing in the clinic, as well as the prospect of manufacturing a synthetic monoclonal anti-Vel antibody to be used in diagnostics, blood group typing and research. Key words Blood groups, Vel blood group system, genetic variation, transcriptional regulation Classification system and/or index terms (if any) Supplementary bibliographical information Language English ISSN and key title 1652-8220 ISBN 978-91-7619-428-7 Recipient’s notes Number of pages 94 Price Security classification I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation. Signature Date February 22nd 2017 Identification and characterisation of SMIM1 variants determining the Vel blood group Mikael Kronborg Christophersen © Mikael Kronborg Christophersen Faculty of Medicine Department of Laboratory Medicine Division of Hematology and Transfusion Medicine Cover photos by Camilla Nehammer ISSN 1652-8220 ISBN 978-91-7619-428-7 Lund University, Faculty of Medicine Doctoral Dissertation Series 2017:48 Printed in Sweden by Media-Tryck, Lund University Lund 2017 Yet an experiment, were you to try it, could free you from your cavil, and the source of your arts' course springs from experiment. -Dante Alighieri: The Divine Comedy Preface The following PhD thesis details the results obtained through four years of research at the Division of Haematology and Transfusion Medicine, Lund University. It consists of three studies focusing on the Vel blood group system and one studying the disease multiple myeloma. Three studies have been published in Nature Genetics, Scientific Reports and Blood and one is an almost submission- ready manuscript. The Vel blood group antigen is present on red blood cells from all humans except rare Vel-negative individuals who can form antibodies to Vel in response to transfusion or pregnancy. It was first described in 1952 as a high incidence antigen, while the molecular background was recently discovered to be a 17-bp deletion in Small Integral Membrane Protein 1, that causes a frame-shift mutation and abolishes SMIM1 expression, thus creating a Vel-negative phenotype. This thesis contains one of the three original discovery studies reporting the Vel antigen-defining SMIM1-deletion. We analysed SNP microarray data from Vel- positive and -negative individuals and identified SMIM1 as a potential gene. We then found the 17-bp deletion to only be present in Vel-negative individuals and sought to finally prove SMIM1 as the genetic background for the Vel antigen by examining: 1) SMIM1 mRNA sequence and expression levels, 2) presence of SMIM1 and the Vel antigen in red blood cell membranes from Vel-positive and - negative individuals and 3) anti-Vel antibody reactivity in red blood cells from individuals homozygous or heterozygous for the 17-bp deletion and in erythroleukaemia cells expressing wild type and mutant SMIM1 protein. Our discovery allowed Vel to be officially recognised by the International Society of Blood Transfusion as blood group system 034. The SMIM1 deletion is the major determinant for Vel expression, yet even Vel- positive individuals show substantial variation in reactivity with anti-Vel antibody, creating a risk for Vel blood typing errors and transfusion reactions. We suspected the cause to be sequence variants in SMIM1 and found rs143702418 (insertion, C>CGCA) and rs1175550 (A>G) to independently influence expression of SMIM1, potentially mediated by the erythroid transcription factor TAL1 that binds preferentially to the high-expressing rs1175550G allele. Lastly, we examined historical Vel-negative samples and sought to retroactively reconcile historic Vel designations to our current knowledge of the SMIM1 deletion variant. As such, we found the old Vel;–1,–2 designation, attributed to individuals phenotyped to have weak to no Vel antigen expression, to correspond to homozygosity for the SMIM1 deletion, while persons designated Vel;1,–2, i.e. low Vel antigen expression, matched with being heterozygous carrier of the deletion. This thesis assisted in characterising the molecular background of the Vel antigen, which paved the way for molecular Vel typing in the clinic as well as the prospect of manufacturing a synthetic monoclonal anti-Vel antibody to be used in diagnostics, blood group typing and research. The final Study IV details the discovery of robust biomarkers for improved FACS sorting of multiple myeloma plasma cells in a bone marrow sample, a project I was involved in during the first year of my PhD. The thesis is divided into four main sections: An introduction outlining haematopoiesis, erythroid transcription factors, blood group systems