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.