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Genetic Background of the Rare Yus and Gerbich Blood Group Phenotypes: Homologous Regions of the GYPC Gene Contribute to Deletion Alleles

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Genetic Background of the Rare Yus and Gerbich Blood Group Phenotypes: Homologous Regions of the GYPC Gene Contribute to Deletion Alleles research paper Genetic background of the rare Yus and Gerbich blood group phenotypes: homologous regions of the GYPC gene contribute to deletion alleles Elise Gourri,1 Gregory A. Denomme,2 Summary Yvonne Merki,1 Erwin A. Scharberg,3 The GYPC gene encodes the glycophorins C and D. The two moieties Cedric Vrignaud,4,5,6 Beat M. Frey,1 Thierry Peyrard4,5,6 express 12 known antigens of the Gerbich blood group system and and Christoph Gassner1 functionally stabilize red blood cell membranes through their intracellular 1Department of Molecular Diagnostics and interaction with protein 4.1 and p55. Three GYPC exon deletions are Research & Development, Blood Transfusion responsible for the lack of the high-frequency antigens Ge2 (Yus type, exon Service Zurich,€ Swiss Red Cross (SRC), 2 deletion), Ge2 and Ge3 (Gerbich type, exon 3 deletion), and Ge2 to 4 Zurich-Schlieren,€ Switzerland, 2Diagnostic (Leach type, exons 3 and 4 deletion), but lack exact molecular descrip- Laboratories and Blood Research Institute, Blood tion. A total of 29 rare blood samples with Yus (GE:-2,3,4) and Gerbich Center of Wisconsin, Milwaukee, WI, USA, (GE:-2,-3,4) phenotypes, including individuals of Middle-Eastern, North- 3 Institute of Transfusion Medicine and African or Balkan ancestry were examined genetically. All phenotypes could Immunohematology, Baden-Baden, German Red be explained by 4 different Yus alleles, characterized by deletions of exon 2 Cross Blood Service Baden-Wurttemberg€ - and adjacent introns, and 3 different Gerbich alleles, with deletions of exon Hessen, Baden-Baden, Germany, 4Departement 3 and adjacent introns. A 3600 base pair GYPC region, encompassing exon Centre National de Reference pour les Groupes Sanguins, Institut National de la Transfusion 2 and flanking region, shares a high degree of sequence homology with a Sanguine (INTS), 5Inserm UMR_S1134, and region flanking exon 3, probably representing an evolutionary duplication 6Laboratoire d’Excellence GR-Ex, Paris, France event. Defining the expression of Gerbich variants presently relies on rare serological reagents. Our approach substitutes the serological characteriza- Received 27 October 2016; accepted for tion with a precise genotype approach to identify the rare Yus and Gerbich publication 11 December 2016 alleles. Correspondence: Christoph Gassner, Molecular Diagnostics and Research & Development Keywords: blood groups, red cell antigens, immunohaematology, (MOC), Blood Transfusion Service, SRC, transfusion medicine, immunogenetics, molecular genetics. Zurich,€ Rutistr. 19, 8952-Schlieren, Switzerland. E-mail: [email protected] EG and GD contributed equally to this work. TP and CG also share equal responsibility for the study. Antigens of the Gerbich blood group system are the result of The GYPC gene is comprised of 4 exons and spans approxi- the expression of the integral membrane sialoglycoproteins mately 48 kilobases on the long arm of chromosome 2. glycophorin C (GPC) and glycophorin D (GPD). These gly- Unlike multi-cistronic blood group system loci, like RHD/ cophorins are important in the maintenance of the red blood RHCE and GYPA/GYPB/GYPE that are subject to a signifi- cell (RBC) plasma membrane structure through their interac- cant antigen diversity due to intergenic recombination events tion with protein 4.1 and p55, which links GPC and GPD to (Wagner & Flegel, 2014; Meyer et al, 2016), GYPC has no spectrin and actin (Alloisio et al, 1993; Marfatia et al, 1994). homologous gene and it shares little similarity with GYPA, Both proteins result from the expression of one single gene, GYPB and GYPE. It shows an alternate start codon resulting GYPC (Le Van Kim et al, 1987), with GPD representing a in the expression of GPD sialoglycoproteins (Cartron et al, 21 N-terminal amino acid truncated version of GPC and 1990). Exons 2 and 3 of GYPC and their flanking regions of encoded through an alternate transcription mechanism (Tan- approximately 3600 base pairs share 95% identity (Fig 1), ner, 1988; El-Maliki et al, 1989; Le Van Kim et al, 1996). suggesting that the exon organization originally arose from a ª 2017 John Wiley & Sons Ltd, British Journal of Haematology doi: 10.1111/bjh.14578 E. Gourri et al Fig 1. Schematic representation of GYPC and its two homologous repeat regions (light grey and black blocks). The deletions found in the Yus and Gerbich alleles are depicted as dashed lines. Diagnostic PCR-SSP products of wild-type-reactions are shown as dark grey blocks and numbered 1–8. Diagnostic gap PCRs are indicated as numbers 9–12 and 13–15, for the four Yus and three Gerbich alleles, respectively. Locations of primers for diagnostic PCRs are indicated as arrows. duplication event. Intragenic recombination events result in 2005). The mechanism of this anaemia is similar to the one nucleotide deletions encompassing one or more exons and, observed with antibodies to the Kell antigens (Denomme sometimes, their flanking regions. These regions of identity et al, 2006). In vitro studies have shown that antibodies to misalign during meiosis to create new alleles and conse- glycophorin C inhibit growth of early erythroid progenitor quently lead to new variant proteins (for a review, see cells by inducing mitochondrial depolarization and per- Daniels, 2013). turbing intracellular phosphorylation pathways; this leads to The Gerbich blood group system [International Society of actin polymerization and the induction of caspase-indepen- Blood Transfusion (ISBT) 020] is comprised of 12 antigens; dent apoptosis (Micieli et al, 2010). This is consistent with 5 low-frequency and 7 high-frequency antigens (International the early expression of glycophorin C on erythroid progeni- Society of Blood Transfusion, 2008). There are at least 3 tor cells during ontogeny. As a result, during pregnancy and variants with affected GPC expression and with or without postpartum, Gerbich antibodies not only cause classical co-expression of GPD that result in the loss of some high- macrophage-dependent erythroid cell destruction, but also prevalence antigens (Walker & Reid, 2010). The Yus (Yussef, inhibit early erythroid progenitor cell proliferation. The GE:-2,3,4) and Gerbich (GE:-2,-3,4) phenotypes are the result absence of reticulocytosis in haemolytic disease of the fetus of a deletion of exon 2 (GE*01.-02) and exon 3 (GE*01.-03) and newborn due to Gerbich maternal antibodies is consis- respectively. Another exceptional form, the Leach phenotype tent with the effect on erythroid cell proliferation (Arndt (GE:-2,-3,-4) with a complete absence of all GPC and GPD et al, 2005). proteins, is due to the absence of exons 3 and 4 (GE*01N.01) The anti-Gerbich antibodies are potentially clinically rele- or a missense mutation in the exon 3 (GE*01N.02) (Interna- vant and can cause haemolytic transfusion reactions (Baughn tional Society of Blood Transfusion, 2008; Daniels, 2013). et al, 2011). Alloimmunization among persons with the Yus The malarial merozoite form of Plasmodium falciparum uses and Gerbich phenotypes is complex. It appears that some GPC and GPD as a receptor to invade human RBCs (Patel individuals of the Gerbich phenotype make anti-Ge3 without et al, 2001; Mayer et al, 2002). Therefore, variants that alter anti-Ge2. Furthermore, the Gerbich phenotype (GE:-2,-3,4) the expression of exofacial domain probably arose from the seems to present with predominantly anti-Ge2 or anti-Ge3. natural selective pressure of the negative health effects of However, antibodies found in the Yus phenotype (GE:-2,3,4) malaria (Maier et al, 2003). Worldwide epidemiological stud- consistently show an anti-Ge2 specificity. The antibodies to ies have shown that variants of the Gerbich blood group sys- the high-prevalence antigens are the most challenging to tem are observed in two major areas, Middle East/North characterize. For Gerbich, no commercial antisera are avail- Africa/Balkans, and Southeast Asia/Indonesia/Papua New able and antibody identification relies on rare red cells and Guinea. P. falciparum infestations are common in both of alloantibodies shared among investigators. Sometimes these these areas (Mendis et al, 2001). reagents are not as they appear and errors are made in the Antibodies to Gerbich blood group antigens are important classification of new samples. Such antibody studies can ben- to consider in transfusion and pregnancy. The antibodies efit from samples that are well characterized at the molecular may be associated with haemolytic transfusion reactions and level to ensure identity between proband samples. haemolytic disease of the newborn. Gerbich antibodies are The nucleotide sequence homology between exons and particularly of clinical significance in pregnancy because they flanking regions, together with deletion variant alleles, can are often associated with a late-onset anaemia (Arndt et al, make nucleotide interrogation (genotyping) for the prediction 2 ª 2017 John Wiley & Sons Ltd, British Journal of Haematology GYPC Deletion Alleles of blood group antigens a challenge. In addition, variation in using standard reagent red cells, previously characterized Yus the location of the deletion, i.e. the specific breakpoint of the and Gerbich red cells and red cells available from a cryopre- deletion, can place the remaining information in a different served collection. context for mRNA processing and expression. We used a col- lection of alloimmunized individuals of the Yus and Gerbich Positional PCR, Sanger sequencing, and diagnostic phenotypes to characterize their nucleotide breakpoint regions PCR-SSP
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