UvA-DARE (Digital Academic Repository) Genetic basis of rare blood group variants Wigman, Lonneke. Link to publication

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1 UvA-DARE (Digital Academic Repository) Genetic basis of rare blood group variants Wigman, Lonneke 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: 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 ( Download date: 11 Oct 2018

2 Chapter 1 General introduction and scope of this thesis

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4 General introduction Red blood cells carry and deliver oxygen to all cells of our body and are therefore essential for human life. During trauma huge amounts of red blood cells can flow out of the body. A massive loss of red blood cells will lead to the failure of oxygen transport to vital organs, such as the heart and brain, resulting in death. Furthermore, some individuals have a defect in erythropoiesis (for instance thalassaemia) which can cause constitutional anemia. To relieve massive blood loss after trauma or to relieve anemia in patients with an erythropoiesis disorder, red blood cells from a donor can be transfused. Donor blood cannot be simply transfused to every patient in need of transfusion. On the surface of red blood cells blood group antigens are present. When a recipient of a red blood cell transfusion has an antibody to a blood group antigen present on the transfused donor red blood cells, the immune system of the recipient can destroy all donor red blood cells, which results in a sometimes fatal transfusion reaction. Consequently, it is very important to transfuse compatible donor red blood cells to an immunized recipient. Chapter 1 Blood group antigens In 1901, Landsteiner discovered that red blood cells of different healthy humans were not completely the same. 1 He detected three differences on the membrane of red blood cells and named them A, B and O. 1 Although intensive research was done to detect more blood group antigens, it took more than 25 years to discover another blood group antigen, the M antigen. 2-4 The majority of blood group antigens were discovered after the development of the indirect antiglobulin test by Coombs et al. in 1945, which dramatically increases the sensitivity of serological blood group typing. 2,5 At this moment more than 300 different blood group antigens are known, which are divided into thirty-three blood group systems, six blood group collections and two red blood cell antigen series (Table 1). 6,7 It is expected that the Vel blood group, of which the genetic basis was recently elucidated, will be assigned as the 34 blood group system (Table 1). Blood group antigens are structures, proteins, carbohydrates or lipids, present on the membrane of red blood cells (Figure 1). 6,8,9 Not all structures on the red blood cell membrane are blood group antigens. 6,8,9 To be entitled as a blood group antigen, the antigen must be serologically characterized by human antibodies and the antigen must be inheritable. 6,8,9 Origin of blood group antigens The huge variety of blood group antigens is thought to have arisen due to evolutionary pressure of various pathogens. 10,11 The different antigens of the carbohydrate blood groups are assumed to originate from pressure of microbial pathogens. 12,13 Parasitic pathogens such as malaria are responsible for differential expression of blood group antigens carried on protein 9

5 Chapter 1 structures. 14,15 The effect of malaria on blood group antigen expression can still be observed via the geographic distribution of specific blood group phenotypes For instance, in West- Africa almost 100% of the population lacks the complete expression of the Duffy glycoprotein, which makes them resistant for malaria infection by Plasmodium vivax. 14,16 Nevertheless, for many blood group antigens the evolutionary pressure is not known and possibly even non existing. Differential blood group antigen expression might also be due to genetic drift, for many blood group antigens the geographic distribution can simply be explained by founder effects. 17 Figure 1. Model of structures that carry blood group antigens on the red blood cell membrane. (modified from Reid et al. 2013; ref 6 ). Molecular basis of blood group antigens The first blood group antigens for which the molecular basis was clarified were the M and N antigen, when Siebert and Fukuda cloned the GYPA gene in Due to the rapid evolvement of DNA techniques, the genetic basis of the majority of blood group antigens was elucidated in less than ten years. 10,11,19 At this moment there are, however, still blood group antigens of which the genetic basis is not elucidated, for instance the At a and Emm blood group antigens. 6,9 Differential expression of blood group antigens can be due to single 10

6 General introduction Table 1. Overview of the 34 blood group systems, 6 blood group collections and 2 red blood cell antigen series ISBT No ISBT name Number of antigens Blood group systems Chromosome location Gene Antigen 1 Frequency of antigen 1* Genetic difference(s) between antigen 1 and antigen 2 Antigen 2 Frequency of antigen 2* 1 ABO 4 9q34.1-q34.2 ABO A 47% c.297a>g c.526c>g c.657c>t c.703g>a c.796 C>A c.803 G>C c.930 G>A B 13% 2 MNS 46 4q31.21 GYPA M 78% c.59c>t c.71g>a c.72t>g N 72% GYPB S 55% c.143c>t S 89% 3 P1PK 3 22q13.2 A4GALT P 1 79% c.42c>t P 2 21% 4 Rh 52 1p36.11 RHD D 83% Deletion of RHD Null 17% phenotype RHCE C 68% c.48c>g c.178a>c c.201g>a c.203g>a c.307 c 80% T>C E 29% c.676c>g e 98% 5 Lutheran 20 19q13.32 LU Lu a 8% c.230g>a Lu b 99.8% 6 Kell 34 7q34 KEL K 9% c.578c>t k 99.8% Kp a 2% c.841c>t Kp b 100% 7 Lewis 6 19p13.3 FUT3 Le a 22% Le b 72% 8 Duffy 5 1q23.2 DARC Fy a 66% c.125g>a Fy b 83% 9 Kidd 3 18q12.3 SLC14A1 Jk a 77% c.838g>a Jk b 74% 10 Diego 22 17q21.31 SLC4A1 Di a 0.01% c.2561c>t Di b 100% Wr a <0.01% c.1972g>a Wr b 100% 11 Yt 2 7q22.1 ACHE Yt a >99.8% c.1057c>a Yt b 8% 12 Xg 2 Xp22.33 PBDX Xg a 89% / Not determined 66%** MIC2 CD99 100% Not determined Null Rare phenotype 13 Scianna 7 1p34.2 ERMAP Sc1 >99% c.169g>a Sc2 1% 14 Dombrock 8 12p12.3 ART4 Do a 67% c.378c>t c.624t>c c.793g>a Do b 82% Chapter 1 11

7 Chapter 1 Table 1. (Continued) ISBT No ISBT name Number of antigens Chromosome location Gene Antigen 1 Frequency of antigen 1* Genetic difference(s) between antigen 1 and antigen 2 Antigen 2 Frequency of antigen 2* 15 Colton 4 7p14.3 AQP1 Co a 99.5% c.134c>t Co b 10% 16 Landsteiner- Wiener 17 Chido- Rodgers 3 19p13.2 ICAM4 LW a 100% c.299a>g LW b <1% 9 6p21.32 C4B Ch1 96% c.3620c>t c.3629g>t c.3630g>c C4A Rg1 >98% c.3567a>g c.3660t>c c.3669t>g c.3670c>g R 18 Hh 1 19q13.33 FUT1 H >99.9% c.422g>a Null phenotype 19 Kx 1 Xp21.1 XK K x 100% Deletion of XK Null phenotype 20 Gerbich 11 2q14.3 GYPC Ge2 >99.9% Deletion of exon 2 of GYPC Ge-2 Rare Rare Rare Ge3 >99.9% Deletion of exon 3 of GYPC Ge-2-3 Rare 21 Cromer 16 1q32.2 DAF Cr a 100% c.679g>c Cr a- Rare 22 Knops 9 1q32.2 CR1 Kn a 94.5% c.4681g>a Kn b 3.5% 23 Indian 4 11p13 CD44 In a 0.10% c.137g>c In b 99% 24 Ok 3 19p13.3 BSG Ok a 100% c.274g>a Ok a- Rare 25 RAPH 1 11p15.5 CD151 MER2 92% c.511c>t Mer- 26 JMH 6 15q24.1 CD108 JMH 100% c.619c>t JMHK- Rare 27 I 1 6p24.2 GCNT2 I >99% c.1049g>a Null phenotype 28 GLOB 1 3q26.1 B3GALNT1 P >99.9% c.202c>t Null phenotype 29 Gill 1 9p13.3 AQP3 GIL 100% c.ivs5+1g>a Null phenotype 30 RHAG 4 6p21.3 RHAG Duclos 100% c.316g>a Duclos- Rare <1% <0.01% Rare 31 FORS 1 9q34.2 GBGT1 Fors1 <0.1% c.887g>a Fors1- >99.9% 32 JR 1 4q22.1 ABCG2 Jr a 99.9% c.376c>t Null phenotype <0.1% 12

8 General introduction Table 1. (Continued) ISBT No ISBT name Number of antigens Chromosome location Gene Antigen 1 Frequency of antigen 1* Genetic difference(s) between antigen 1 and antigen 2 Antigen 2 Frequency of antigen 2* 33 LAN 1 2q36 ABCB6 Lan >99.9% c.197_198insc Null phenotype 34w Vel 2 1p36.32 SMIM1 Vel >99.9% c.64_80del Null phenotype Blood group collections <0.01% <0.1% 205 Cost 2 Cs a >98% Cs b 34% 207 Ii 1 i 208 Er 3 Er a 100% Er b <0.01% 209 Globoside 2 LKE 98% 210 unnamed 2 Le c 1% 213 MN CHO 6 Hu 1% Red blood cell antigen series 700 Low- Incidence antigens 900 High- Incidence antigens 18 By Rare 6 At a 100% * When the frequency differs between populations, the frequency of the Caucasian population is stated Silent mutations Multiple mutation can each cause the phenotype of antigen 2, the most frequently occuring mutation is stated The presense or absence of the Le a and Le b antigens is determined by the functionality of multiple fucosyltransferases encoded by the FUT gene family ** Differential expression between females and males, respectively Most likely the frequency of the RAPH antigen is higher, the expression is, however, in many persons to low to detect with standard serological typing Chapter 1 13

9 Chapter 1 nucleotide polymorphisms, multiple nucleotide polymorphisms or genetic rearrangements (Table 1). 10,19 Described below are three blood group systems, the ABO, Rh and Kell blood group systems, that provide an example for each of the three genetic mechanisms. The ABO blood group system The antigens of the ABO systems are carbohydrate structures. 20,21 The difference between the A and B antigen is the presence of respectively a N-acetyl-D-galactosamine or a D-galactose at the end of a carbohydrate carrier structure. 22,23 Individuals who have the O antigen lack the presence of any carbohydrate at this position. 24 The ABO gene that is responsible for the expression of the ABO antigens, codes for an enzyme of the glycosyltransferase cluster. Glycosyltransferases attach carbohydrates to an oligosaccharide acceptor chain. 25 Individuals with blood group A have an allele of ABO coding for a1,3-n-acetylgalactosaminyltransferase [A transferase] that can only attach the N-acetyl-galactosamine carbohydrate. 26 Individuals with blood group B have an allele that codes for the highly similar a1,3-galactosyltransferase [B transferase], but this enzyme can only attach the galactose carbohydrate. 27 The difference in enzymatic specificity between the A and B transferases is due to four amino acid changes caused by four nucleotide polymorphisms in ABO (Table 1). 28 Individuals who have the O blood group have a mutated allele that codes for a non-functional protein and therefore lack the expression of A or B transferase. 21,29 Many different alleles of the ABO gene are known that give rise to the O antigen, the most frequently occurring O allele has a deletion of one single nucleotide. 21,29 The Rh blood group system The Rh blood group system is encoded by two very homologous genes, RHD and RHCE, that code for the RhD and RhCE protein respectively Individuals who are D negative [D-] lack the expression of the RhD protein. 32,33 In the Caucasian population 17% of the population is D-, which is almost always caused by the complete deletion of RHD (Table 1). 6,32,33 In the Negroid population 8% of the population is D-, in this population the D- phenotype is in 19% due to a complete deletion of RHD, in 66% due to the RHD*Ψ allele (RHD*Pseudogene) with a 37 base pair insertion and a premature stopcodon and in 15% due to the hybrid RHD*03N.01 allele. 6,34-36 In the Asian population the D- phenotype is very rare less than 0.5% of the population is D-. 6,37 Next to the normal D positive [D+] and D- expression, three types of variant RhD expression exist: weak D, D el and partial D expression. 6,9,36 Individuals with weak D expression express the complete RhD protein, however, in low quantities; between 100 to 5000 RhD antigens per red blood cell compared to the 10,000 till 30,000 antigens in normal D+ individuals. 6,9,36 Weak D expression is most often caused by single mutations in RHD that cause an amino acid change in the transmembrane or intracellular parts of the protein. 38 The D el phenotype is also characterized by weak expression of the complete RhD protein. However, individuals with the 14

10 General introduction D el phenotype have such low expression of the RhD protein that expression is only detectable with the very sensitive absorption-elution technique, to which the name of this phenotype refers: D-elution. 39 The D el phenotype is most often caused by mutations that disturb splice sites or mutations in the C-terminal region of the RhD protein. 37,40-42 Individuals with a partial D variant express an RhD protein that lacks one or several of the thirty D-epitopes Most partial variants arise from hybrid alleles in which parts of RHD are exchanged with the very homologous RHCE gene These hybrid alleles encode a correctly folded protein that lacks certain D-epitopes because the CE counterparts are present at these positions Partial D variants can also arise due to single or multiple mutations that cause amino acid changes in (an) extracellular loop(s) of the RhD protein In many partial D variants the expression is also weakened compared to normal RhD expression. 9 Chapter 1 The Kell blood group system The antigens of the Kell blood group system are also carried on a protein, which is encoded by the KEL gene However, in contrast to RhD, the differential expression between for instance the K and its antithetical k antigen is due to the difference of a single nucleotide polymorphism that causes a single amino acid difference (Table 1). 54 For other Kell blood group antigens, for instance the Kp a and its antithetical Kp b antigen, the differential expression is also caused by a single amino acid change, but at a different position on the Kell protein compared to the K/k antigens (Table 1). 55 The complete lack of the Kell protein, the Kell null phenotype, is very rare This phenotype is caused by a heterogeneous set of mutations resulting in either a premature stopcodon or disruption of a splice site in KEL. 6,59-61 High frequency blood group antigens High frequency blood group antigens are blood group antigens that are expressed in more than 90% of the individuals of most populations. 6,8,9 For patients with antibodies to high frequency antigens it is difficult to obtain compatible donor red blood cells in a short time frame. 9,62 The low occurrence of individuals who are negative for a high frequency blood group antigen, for instance 0.04% of the population is Vel- and only 0.005% of the population is Lan-, makes it difficult to obtain large numbers of donors who are negative for a high frequency antigen. 6,9 Furthermore, because for most high frequency antigens reliable typing reagents are lacking, the donor population is not routinely typed to identify donors that are negative for a high frequency antigen. To overcome shortage of donor blood negative for a high frequency antigen this donor blood is frozen and stored, for instance, in the Sanquin Bank of Frozen Blood in the Netherlands, until a patient is in need of the blood. 63 Disadvantages of frozen blood are the high costs of freezing and maintaining frozen red blood cells and the large amount of time it takes before the red blood cells can be transfused into a patient, because the red blood cells need to be thawed, washed and shipped to the patient

11 Chapter 1 Little was known about the high frequency antigens Vel, Jr a and Lan at the start of this project. 9 All three are not expressed on granulocytes, monocytes or lymphocyte and all three antigens are resistant to enzymatic and chemical treatments of red blood cells using for instance bromelain, chymotrypsin, DTT or acid. 6,65,66 The Vel antigen The Vel antigen was first described in 1952 in a patient who suffered from an acute hemolytic transfusion reaction after a blood transfusion. 67 The identified antibody was directed against a high frequency antigen, subsequently named Vel. 67 Multiple severe hemolytic transfusion reactions in patients with anti-vel have since been described A large individual variation in the level of Vel expression is present and in some people the Vel expression is very weak, making it difficult to correctly type for the presence and absence of the Vel antigen. 72 The Jr a and Lan antigens The first examples of respectively the Jr(a-) and the Lan- phenotype were described at congresses in 1970 and ,74 Both anti-jr a and anti-lan are able to induce a severe hemolytic transfusion reaction to Jr(a+) or Lan+ red blood cells, respectively. 73,75 The capacity of anti-jr a to destroy red blood cells seems to increase after each encounter with the Jr a antigen. 75,76 Moreover, hemolytic transfusion reactions due to anti-jr a seem to get more severe after each incompatible blood transfusion and also hemolytic disease of the fetus and newborn was getting more severe in subsequent pregnancies of a woman with anti-jr a. 75,76 Recently, the genetic basis of the Jr(a-) phenotype and the Lan- phenotype were elucidated The Jr a and Lan antigens are both carried on a protein structure, the ABCG2 or ABCB6 protein, respectively, and negativity is caused by the complete absence of the protein Multiple mutations have been described in ABCG2 or ABCB6 that can each cause negativity for the Jr a or Lan antigen, respectively The Jr(a-) phenotype is most often caused by the c.376c>t, c.706c>t or c.736c>t mutation in ABCG2 which all introduce a premature stopcodon. 77,78,80 The molecular basis of Lan- shows large heterogeneity, in 49 Lan- persons a total of 24 different mutations are described. 79,82 Only three mutations, c.459del, c.574c>t and c g>a in ABCB6, have been detected in five or more Lan- individuals. 82 Before the genetic basis of the Jr a and Lan antigens were linked to the ABCG2 and ABCB6 protein, respectively, these two proteins were already studied for their putative porphyrin transporter function. 83 In individuals who are Jr(a-) or Lan- porphyrin levels are elevated, confirming the function of ABCG2 and ABCB6 as porphyrin transporter. 77,79 Heme is a porphyrin and ABCG2 and ABCB6 may play a role in heme uptake into the red blood cell. No aberrant red cell parameters were, however, detected in Jr(a-) and Lan- individuals. 77,79 Allo-antibodies to blood group antigens 16

12 General introduction Allo-antibodies are antibodies in individuals who themselves lack the corresponding antigen. 9,64 Allo-antibodies can be naturally occurring or immune antibodies. 6,9,64 The ABO blood group antigens have naturally occurring allo-antibodies. 6,9,21 Soon after birth everybody with, for instance, blood group A will develop antibodies to blood group B they themselves lack. 6,9,21 Immune allo-antibodies only arise after an encounter with a blood group antigen that an individual lacks on his/her own red blood cells. 6,9 These foreign antigens can be encountered during a transfusion of red blood cells or during the fetal-maternal blood contact in a pregnant woman or during delivery When an individual encounters red blood cells carrying foreign antigens, they will be cleared normally by the spleen Antigens from the cleared red blood cells are processed and peptides from the antigens are presented to the immune system via HLA class II. 90,91 When the immune system detects a peptide from a foreign antigen an immune response occurs and allo-antibodies are produced to the antigen. 89,92 Chapter 1 Clinical implications of blood group allo-antibodies Allo-antibodies are clinically important, because they are able to destroy red cells that are positive for the corresponding antigen. 64,93,94 Allo-antibodies play a role in two kinds of hemolytic reactions: hemolytic transfusion reaction [HTR] and hemolytic disease of the fetus and newborn [HDFN]. 64,93,94 Hemolytic transfusion reaction Although transfusion of red blood cells is generally very safe, HTRs remain an important transfusion hazard. 95,96 An HTR occurs when an individual is transfused with red blood cells positive for an antigen to which he/she has made antibodies. 64,93,94 During an HTR the transfused red blood cell are destroyed via intravascular or extravascular hemolysis. 64,93,94 Intravascular hemolysis is characterized by the destruction of the red blood cells in the bloodstream due to the activation of the complete complement cascade. 97,98 The red blood cells are instantly destroyed and the cytotoxic hemoglobin is released into the bloodstream, which in severe cases can be fatal Not all allo-antibodies are able to cause intravascular hemolysis, alloantibodies to the ABO blood group antigens and the Vel antigen are known offenders of intravascular hemolysis. 93 Extravascular hemolysis is characterized by the enhanced clearance of red blood cells via phagocytosis by macrophages in the spleen and the liver. 97,98 The main complications of extravascular hemolysis are the same as in intravascular hemolysis, the symptoms are, however, generally less severe. 94 Extravascular hemolysis involves alloantibodies that are not able to completely activate the complement system, but the alloantibodies prime the red blood cells for enhanced clearance. 93 Hemolytic disease of the fetus and newborn HDFN is a disease in which the red blood cells of the fetus and/or newborn are destroyed 17

13 Chapter 1 by allo-antibodies from the mother. 86,100,101 Antibodies of a pregnant woman including alloantibodies to red blood cell antigens are able to cross the maternal/fetal placental barrier and when the red blood cells of the fetus are positive for the corresponding antigen they will be destroyed due to the maternal antibodies. 102 In severe cases of HDFN the fetus becomes anemic due to the large destruction of his/her red blood cells. 86 Anemia in the fetus can only be treated with intra-uterine transfusions. 86 Furthermore, mild cases of HDFN, that do not need intra-uterine transfusion, can become life-threatening after birth. 103 Babies with HDFN have higher bilirubin concentrations, because bilirubin is released into the blood stream when red blood cells are destroyed. 104 Before birth, bilirubin is transferred to the mother who excretes it After birth the newborn s liver is too immature to clear the high levels of bilirubin and deposits of bilirubin may occur in the skin or in the brain nuclei, which can cause brain damage The removal of bilirubin can be stimulated by phototherapy or bilirubin concentrations can be lowered via exchange transfusion. 104 HDFN is most often caused by allo-antibodies to the RhD blood group antigen, moreover anti-d often causes severe HDFN. 101 Since the introduction of the administration of anti-d prophylaxis in D- pregnant woman in 1960 the amount of cases of HDFN due to anti-d is substantially decreased. 107,108 Blood group antigen typing For safe transfusion medicine it is important to correctly characterize blood group antigens in blood donors, recipients of red blood cells and pregnant women. 64,93,94 At this moment blood group typing is most often done via serological typing. 6,9,64 Serological typing of red blood cell recipients and/or donors is, however, not always possible. 109 Firstly, after transfusion or in case of the presence of auto-antibodies or multiple allo-antibodies, serological typing is very difficult and less reliable. 110,111 Secondly, for most blood group antigens, serological typing relies on the availability of polyclonal human antisera, hence for some antigens it is impossible to screen large cohorts of donors due to the scarcity of the sera. 112,113 Furthermore, the available sera are not always able to correctly detect variant antigen expression. 48,112,114 It is important to correctly detect the variant expression of a blood group antigen, because red blood cells with weak antigen expression can immunize recipients and can cause a HTR. 115,116 Moreover, recipients with a partial variant of a blood group antigen also need to be correctly identified, because they are able to produce allo-antibodies to the epitopes they miss. 48,114 In cases in which serology is not feasible, it is possible to determine the blood group antigen status via genotyping. 110,111,117 Several blood group genotyping assays have been developed that are able to determine the phenotype of multiple blood group antigens in one test Genotyping has already been used for screening of rare blood group phenotypes in donors and some blood banks even have started to implement genotyping of blood donors as an alternative to serological typing. 111,113,119,124 18

14 Scope of this thesis Scope of this thesis The research presented in this thesis focuses on the genetic background of rare blood group variants and characterization of the effect of these rare blood group variants on antigen expression. Chapter 1 To examine rare blood group antigens, we first had to be able to detect the presence of common blood group antigens. In recent years several dedicated genotyping assays have been developed to genotype multiple blood group antigens in one test. These tests require expensive dedicated equipment and can only detect a limited number of blood group antigens. We therefore aimed to develop a new assay to genotype for blood group antigens using the multiplex ligation-dependent probe amplification [MLPA] technique (chapter 2). The Rh blood group system has large genetic variety resulting in more than 250 RH variant alleles. Some of these RH variant alleles occur frequently in specific populations, for instance the RHD*06.01 allele in the Caucasian population or the RHCE*01.04 allele in the Black population. Many of these variant alleles have arisen due to genetic rearrangements between RHD and RHCE, which makes characterization of the alleles via Sanger sequencing difficult. We aimed to develop a genotyping technique that can type for the presence of the most frequently occurring RH variant alleles (chapter 3). To prevent D immunization, all Dutch D- pregnant women are offered a genetic test to screen for the presence of a D+ fetus. When a D- pregnant woman carries a variant D allele, this will be recognized by this genetic test. The available data from the screening of ~ serologically D- women per year, gave us the opportunity to determine rare Rh variants with extremely low or absent RhD expression in Dutch D- pregnant women. When new variant D alleles are detected, the effect of the variant allele on the RhD phenotype will be determined, to conclude the clinical relevance of these rare variants (chapter 4). Persons immunized to the Vel antigen might experience a severe hemolytic transfusion reaction when transfused with Vel+ red blood cells. Therefore they should only receive very rare Vel- red blood cells. Characterization of Vel- donors via serology is very difficult due the variable expression levels of the Vel antigen. Via genotyping, negativity for the Vel antigen could be verified, however, at the start of this thesis the genetic basis of the Vel- phenotype was not yet elucidated. In chapter 5 it is described how we have determined the genetic background of the Vel antigen and the genetic variation that leads to the Vel- phenotype. As stated above characterization of the Vel- phenotype via serology is very difficult due to the variable levels of Vel expression. We next intended to characterize the effect of mutations 19

15 Chapter 1 and polymorphisms in SMIM1 on the level of Vel expression. Furthermore, a high-throughput genotyping assay was developed to overcome all technical difficulties of serological identification of Vel- donors (chapter 6). For the high frequency antigens Jr a and Lan antigens it is also very difficult to obtain large numbers of negative donors. Due to the rarity of the Jr(a-) and Lan- phenotype and serological impairments, Jr(a-) and Lan- red blood cells are only available in frozen stocks. In chapter 7 we investigated the genetic basis of the Jr(a-) and Lan- phenotype in the Dutch population and we aimed to develop a high throughput screening assay for the detection of Jr(a-) and Lan- donors. Dominant familial azotemia is a very rare disease, which has only been described in two families. The Kidd blood group antigen is carried on a urea transporter, which is also expressed on kidney cells. The Kidd null phenotype is linked with impaired urea homeostasis. In chapter 8 we investigated the genetic background and aberrant urea transport in a Dutch family with dominant familial azotemia. Finally, in chapter 9 a discussion of all results described in this thesis and of blood group genotyping in general is given. 20

16 General introduction References 1. Landsteiner K. On agglutination of normal human blood. Transfusion 1961;1: Daniels G, Reid ME. Blood groups: the past 50 years. Transfusion 2010;50(2): Landsteiner K, Levine P. A new agglutinable factor differentiating individual human bloods. Proc.Soc.Exp.Biol. 1927;24: Landsteiner K, Levine P. Further observations on individual differences of human blood. Proc.Soc.Exp.Biol. 1927;24: Coombs RR, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br.J.Exp. Pathol. 1945;26: Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen Facts Book. 3 ed. San Diego: Academic Press; Storry JR, Castilho L, Daniels G, Flegel WA, Garratty G, Francis CL, Moulds JM, Moulds JJ, Olsson ML, Poole J, et al. International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Berlin report. Vox Sang. 2011;101(1): Lewis M, Anstee DJ, Bird GWG, Brodheim E, Cartron JP, Contreras M, Crookston MC, Dahr W, Daniels G, Engelfriet CP, et al. Blood group terminology The ISBT Working Party on Terminology for Red Cell Surface Antigens. Vox Sang. 1990;58(2): Daniels G. Human Blood Groups. 2 ed. Oxford: Blackwell Science; Storry JR, Olsson ML. Genetic basis of blood group diversity. Br.J.Haematol. 2004;126(6): Denomme GA. Molecular basis of blood group expression. Transfus.Apher.Sci. 2011;44(1): Stowell SR, Arthur CM, Dias-Baruffi M, Rodrigues LC, Gourdine JP, Heimburg-Molinaro J, Ju T, Molinaro RJ, Rivera-Marrero C, Xia B, et al. Innate immune lectins kill bacteria expressing blood group antigen. Nat.Med. 2010;16(3): Gagneux P, Varki A. Evolutionary considerations in relating oligosaccharide diversity to biological function. Glycobiology 1999;9(8): Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium vivax in blacks. The Duffyblood-group genotype, FyFy. N.Engl.J.Med. 1976;295(6): Zimmerman PA, Ferreira MU, Howes RE, Mercereau-Puijalon O. Red blood cell polymorphism and susceptibility to Plasmodium vivax. Adv.Parasitol. 2013;81: Howes RE, Patil AP, Piel FB, Nyangiri OA, Kabaria CW, Gething PW, Zimmerman PA, Barnadas C, Beall CM, Gebremedhin A, et al. The global distribution of the Duffy blood group. Nat.Commun. 2011;2: Kimura M. The neutral theory of molecular evolution: a review of recent evidence. Jpn.J.Genet. 1991;66(4): Siebert PD, Fukuda M. Molecular biological study of the structure and expression of human glycophorin A. Rev. Fr.Transfus.Immunohematol. 1986;29(4): Daniels G. The molecular genetics of blood group polymorphism. Transpl.Immunol. 2005;14(3-4): Yamamoto F. Review: ABO blood group system ABH oligosaccharide antigens, anti-a and anti-b, A and B glycosyltransferases, and ABO genes. Immunohematology. 2004;20(1): Storry JR, Olsson ML. The ABO blood group system revisited: a review and update. Immunohematology. 2009;25(2): Morgan WT, Watkins WM. The inhibition of the haemagglutinins in plant seeds by human blood group substances and simple sugars. Br.J.Exp.Pathol. 1953;34(1): Watkins WM, Morgan WT. Inhibition by simple sugars of enzymes which decompose the blood-group substances. Nature 1955;175(4459): Watkins WM, Morgan WT. Possible genetical pathways for the biosynthesis of blood group mucopolysaccharides. Vox Sang. 1959;4(2): Yamamoto F, Clausen H, White T, Marken J, Hakomori S. Molecular genetic basis of the histo-blood group ABO system. Nature 1990;345(6272): Hearn VM, Smith ZG, Watkins WM. An a-n-acetyl-d-galactosaminyltransferase associated with the human blood-group A character. Biochem.J. 1968;109(2): Chapter 1 21

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