UvA-DARE (Digital Academic Repository) Genetic basis of rare blood group variants Wigman, L. Publication date 2013 Link to publication Citation for published version (APA): Wigman, L. (2013). Genetic basis of rare blood group variants. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:28 Sep 2021 Chapter 6 Genetic screening for the Vel- phenotype circumvents difficult serological screening due to variable Vel expression levels Lonneke Haer-Wigman1 Shabnam Solati1 Aïcha Ait Soussan1 Erik Beckers2 Pim van der Harst3 Marga van Hulst-Sundermeijer4 Peter Ligthart1 Dick van Rhenen5 Hein Schepers3 Tamara Stegmann1 Masja de Haas1 C. Ellen van der Schoot1 1 Sanquin Research, Amsterdam and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands 2 Maastricht University Medical Centre+, Maastricht, The Netherlands 3 University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 4 Sanquin Diagnostic Services, Amsterdam, The Netherlands 5 Sanquin Blood Bank, Rotterdam, Netherlands Manuscript under preparation Chapter 6 Abstract Background: Serological determination of the Vel- phenotype is challenging due to variable Vel expression levels. In this study we investigated the genetic basis for variable Vel expression levels and developed a high-throughput genotyping assay to detect Vel- donors. Methods: In 107 Vel+w and 548 random Caucasian donors genetic variation in the SMIM1 gene was studied and correlated to the level of Vel expression. A total of 3,366 Caucasian, 621 Black and 333 Chinese donors were screened with a high-throughput genotyping assay targeting the SMIM1*64_80del allele. Results: The Vel+w phenotype is caused by the presence of one SMIM1 allele carrying the major allele of the rs1175550 SNP in combination with in most cases a SMIM1*64_80del allele or in few cases the SMIM1*152T>A or SMIM1*152T>G allele. In ~6% of Vel+w donors genetic factors in SMIM1 could not explain the weak expression. We excluded the possibility that lack of expression of another blood group system was correlated with variable Vel expression levels. Furthermore, using a high-throughput Vel genotyping assay we detected two Caucasian Vel- donors. Conclusions: Variable Vel expression levels are influenced by multiple genetic factors in SMIM1 and also by genetic or environmental factors. Due to this variable Vel expression serological typing of the Vel- phenotype is difficult and a genotyping assay targeting the c.64_80del deletion in SMIM1 should be considered to screen donors for the Vel- phenotype. 124 Molecular basis of weak Vel expression Introduction Patients with antibodies directed to the Vel blood group antigen and who are in need of a blood transfusion have a transfusion hazard in an emergency situation.1,2 When a patient with anti-Vel is transfused with Vel+ red blood cells a severe immediate hemolytic transfusion reaction can occur due to intravascular hemolysis of Vel+ red blood cells.3-5 Patients with anti- Vel can therefore only be transfused with Vel- red blood cells, the Vel- phenotype is, however, rare.1,6 The overall frequency of the Vel- phenotype in the Caucasian population is ~0.025% and the Vel- phenotype has been described occasionally in the Asian population and the Chilcotin Indians in Canada.6-9 The availability of Vel- donors is even lower, because donors are not routinely typed for Vel. Serological screening for the Vel- phenotype is cumbersome, due to large individual variation in Vel expression levels and because the scarcely available human anti-Vel sera show heterogeneous levels of reactivity.10 Genetic screening for the Vel- phenotype in the donor population will circumvent the difficult serological typing of the Vel- phenotype. Recently, we and two other groups identified the genetic basis of the Vel antigen and the genetic variation that leads to the Vel- phenotype.11-13 The SMIM1 protein encoded by SMIM1 underlies the Vel antigen and the Vel- phenotype is caused by a 17-base pair deletion in SMIM1 (c.64_80del) that abolishes the complete expression of the SMIM1 protein.11-13 Weak expression of the Vel antigen is correlated with the heterozygous presence of this deletion.11,12 Furthermore, in two individuals with weak Vel expression two single heterozygous missense mutations, at the same nucleotide position of SMIM1, but resulting in a different amino acid substitution, were detected (c.152T>A encoding p.Met51Lys and c.152T>G encoding p.Met51Arg).11 Variation in Vel expression levels may also be related to the rs1175550 variation in intron 2 of SMIM1.11,14 An expression quantitative trait locus [eQTL] study showed that the rs1175550 variation is a regulatory region for SMIM1 transcript levels.11,14 The minor G allele of rs1175550 is associated with higher SMIM1 transcript levels compared to the major A allele.14 This Chapter 6 Chapter regulatory effect of rs1175550 on SMIM1/Vel is supported by the fact that all individuals with weakened Vel expression carried the c.64_80del deletion heterozygous and the major allele of rs1175550 on the non-deletion haplotype.11 The aims of the current study is to characterize the underlying genetic basis of weak Vel [Vel+w] expression and to characterize the effect of the genetic variation of SMIM1 on the expression levels of the Vel antigen. Furthermore, we developed and validated a high-throughput molecular typing assay to identify donors with the Vel- phenotype. 125 Chapter 6 Material and methods Samples and DNA isolation All cases were included after written informed consent was provided. In 2005, 141 donors with weak Vel expression and five Vel- donors were identified upon serological screening of 10,500 donors with a human polyclonal anti-Vel serum. Nineteen of the 141 Vel+w donors have also been reported in previous work.11 For 107 of the 141 donors material collected in 2005 was available. A total of 3,366 Caucasian, 621 Black and 333 Black Asian random donor samples were included. Samples of Dutch Caucasian blood donor samples were collected by Sanquin Blood Supply, The Netherlands. Asian and Black donor samples were collected by The South African Blood Transfusion Service in Johannesburg South-Africa. Additional Black donor samples were collected by The Ethiopian Red Cross Society Transfusion Service in Addis Ababa, Ethiopia and The Red Cross Blood Bank Curaçao in Willemstad, Curaçao. DNA was extracted from white blood cells using a DNA extraction kit (QIAamp DNA Blood Mini Kit). Serology Screening for the Vel- phenotype on 10,500 donors with blood group O was performed using undiluted human serum (NL15) in the Cellbind card technique. All donors with a negative agglutination reaction were tested in a second round using standard tube agglutination technique with NL15 and one to three additional human anti-Vel sera (NL25, NLVzO and/or NLVzA). A donor was determined to have weak Vel expression levels when at least one of the Vel sera gave a positive reaction. Vel antigen expression was examined via agglutination using Liss/Coombs cards (Bio-Rad, Veenendaal, The Netherlands) with anti-Vel serum of NL25 on En(a-), S-s-U-, Rh- (regulator type), In(Lu), Lu(a-b-), K0, Kx-, Fy(a-b-), Jk(a-b-), Gy(a-), Co(a-b-), JMH-, p and Ge:-2,-3 red blood cells and on red blood cells of a patient with paroxysmal nocturnal hemoglobinuria. Agglutination strength was assessed after serum and red blood cells were incubated for 15 minutes at 37ºC with a subsequent spin for 10 minutes. Flow cytometry Vel expression on red blood cells was measured using flow cytometry with immunopurified human polyclonal anti-Vel (NL25 and/or NL04-01). The purified human polyclonal anti-Vel was obtained after incubation of human serum containing anti-Vel with group O Vel+ red blood cells. After washing, the antibodies were eluted from the red blood cells using the Gamma ELU-KIT II (ImmucorGamma). The purified anti-Vel was incubated for 30 minutes at room temperature in a 3% (vol/vol) red blood cell suspension. Subsequently, red blood cells were washed and Alexa-488 labelled goat anti-human-IgG (Molecular Probes) was added in a 1:500 dilution and incubated for 30 minutes at room temperature. Red blood cells were washed and analyzed by flow cytometry (LSRFortessa cell analyzer, Becton Dickinson). Statistical 126 Molecular basis of weak Vel expression analysis was performed using an unpaired two-tailed t-test. The Vel expression levels of the red blood cells of the 548 donors was performed batch wise; the mean fluorescence intensity of the samples was normalized per experiment by correcting the average mean fluorescence intensity per experiment with the average mean fluorescence intensity