Genetic basis of blood group diversity

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1 See discussions, stats, and author profiles for this publication at: Genetic basis of blood group diversity ARTICLE in BRITISH JOURNAL OF HAEMATOLOGY OCTOBER 2004 Impact Factor: 4.71 DOI: /j x Source: PubMed CITATIONS 31 READS 99 2 AUTHORS, INCLUDING: Jill R Storry Lund University 124 PUBLICATIONS 1,236 CITATIONS SEE PROFILE Available from: Jill R Storry Retrieved on: 09 April 2016

2 review Genetic basis of blood group diversity Jill R. Storry and Martin L. Olsson Blood Centre, University Hospital and Department of Transfusion Medicine, Institute of Laboratory Medicine, Lund, Sweden Summary In the last 18 years the genes that encode all but one of the 29 blood group systems present on red blood cells (RBCs) have been identified. This body of knowledge has permitted the application of molecular techniques to characterize the common blood group antigens and to elucidate the background for some of the variant phenotypes. Just as the RBC was used as a model for the biochemical characterization of cell membranes, so the genes encoding blood groups provide a readily accessible model for the study of gene expression and diversity. The application of genotyping techniques to identify fetuses at risk of haemolytic disease of the newborn is now the standard of care, and the expansion of nucleic acid testing platforms to include both disease testing and blood typing in the blood centre is on the horizon. This review summarizes the molecular basis of blood groups and illustrates the mechanisms that generate diversity through specific examples. Keywords: blood group, allele, molecular techniques, genotyping, genetic diversity. For well over a century, blood group antigens have been recognized as differences between the red blood cells (RBCs) of one person and another. Antigens have been defined by human antibodies, immune and naturally occurring, as well as those deliberately stimulated in animals. Assignment of blood group antigen status requires the novel factor to be inherited from one generation to the next, thus demonstrating that blood group antigens were the products of genes. Many of the cellular components that carry blood group antigens have been identified and characterized and indeed, the RBC has provided a useful model for the study of cell membranes. Concurrently, the genes encoding the blood group proteins have been mapped to different chromosomes throughout the genome. The development of DNA sequencing techniques, and then the polymerase chain reaction (PCR) has paved the way for the rapid molecular characterization of the genes encoding blood group antigens, such that, in the last 18 years, all but one of the 29 blood group genes have been characterized. This Correspondence: Martin L. Olsson, Blood Centre, University Hospital and Department of Transfusion Medicine, Institute of Laboratory Medicine, Lund, Sweden. Martin.l_olsson@transfumed.lu.se knowledge, combined with continual improvements in gene analysis are changing the way in which testing can be performed. Blood group antigens are part of carbohydrate or protein structures exposed on the extracellular surface of the RBC membrane. In blood group nomenclature, antigens encoded by the same gene, or cluster of genes, are assigned to the same blood group system. Each system may consist of one or more antigens. Proteins that are glycosylated by N-linked glycans, e.g. band 3 also carry carbohydrate antigens, like ABH and I antigens. Currently, 29 blood group systems, which include a total of 240 antigens, have been established by the International Society of Blood Transfusion (ISBT) Committee on Terminology for Red Cell Surface Antigens. In addition, 38 antigens not yet fulfilling the requirements for classification into a system have been gathered in collections or series of high- and low-frequency antigens (Daniels et al, 2003). These numbers are not static and new blood group antigens are identified by unusual serological findings each year. This review will discuss the genes and polymorphisms underlying the expression of human blood group systems. A comprehensive summary of information detailing the genes and carrier molecule(s) for each blood group system has been collated in Table I and the single nucleotide polymorphisms (SNPs) associated with some clinically important antigens are shown in Table II. References describing the cloning of the genes and the identification of the specific polymorphisms are given in the appropriate table. In addition, a selection of relevant references is given throughout the text, mostly the ones that serve as illustrative examples of different genetic principles. Additional papers describing the elucidation of the polymorphisms responsible for the blood group antigens within each system can be retrieved via the GenBank accession numbers given in Table I and at the Blood Group Antigen Database ( The interested reader is also referred to current textbooks and reviews, a few examples of which can be found in the reference list (Issitt & Anstee, 1998; Reid & Yazdanbakhsh, 1998; Daniels, 2002; Reid & Lomas-Francis, 2003). In general, antigens belonging to blood group systems are better characterized at the molecular level than those antigens assigned to a collection or series. In most instances, the paucity of genetic data regarding the latter antigens prevents assign- ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, doi: /j x

3 Table I. Summary of information on genes and gene products in the currently acknowledged blood group systems. ISBT Name ISBT number ISBT system symbol Chromosome location ISGN gene symbol Genbank account number* Gene product Protein type Apparent mass (kda) Number of amino acidsà CD number Number of antigens Cloning reference ABO 1 ABO 9q34Æ2 ABO NM_ a3galnact, a3galt II n.a Yamamoto et al (1990b)) MNS 2 MNS 4q28Æ2- q31æ1 GYPA NM_ Glycophorin A I CD235a 43 Siebert and Fukuda (1986) GYPB NM_ Glycophorin B I CD235b Siebert and Fukuda (1987) P 3 P1 22q11Æ2-ter P1 a4galt? - n.a. 1 Rh 4 RH 1p36Æ13-p34Æ3 RHD RHCE NM_ NM_ RhD RhCE M-12 M CD240D CD240CE 48 Avent et al (1990), Cherif-Zahar et al (1990), Le Van Kim et al (1992), Arce et al (1993), Kajii et al (1993) Lutheran 5 LU 19q13Æ2 LU NM_ Lutheran glycoprotein (IgSF) I CD Parsons et al (1995) B-CAM (IgSF) I 557 Kell 6 KEL 7q33 KEL NM_ Kell glycoprotein II CD Lee et al (1991) Lewis 7 LE 19p13Æ3 FUT3 NM_ a3/4fuct II n.a Kukowska-Latallo et al (1990) Duffy 8 FY 1q21-q25 DARC NM_ DARC, Duffy glycoprotein M CD234 6 Chaudhuri et al (1993) Kidd 9 JK 18q11-q12 SLC14A1 NM_ HUT, Kidd glycoprotein M Olives et al (1994) Diego 10 DI 17q12-q21 SLC4A1 NM_ AE-1, Band 3 M CD Tanner et al (1988), Lux et al (1989) Yt 11 YT 7q22 ACHE NM_ Acetylcholinesterase GPI Li et al (1991) Xg 12 XG Xp22Æ32 XG NM_ Xg glycoprotein I CD99 2 Darling et al (1986), Ellis et al (1994) Scianna 13 SC 1p34 ERMAP NM_ ERMAP (IgSF) I Ye et al (2000) Dombrock 14 DO 12p13Æ2-p12Æ1 ART4 NM_ ADP-ribosyltransferase 4 GPI Gubin et al (2000) Dombrock glycoprotein Colton 15 CO 7p14 AQP1 NM_ CHIP, Aquaporin-1 M-6 28 or Preston and Agre (1991) Landsteiner- Wiener Chido- Rodgers 16 LW 19p13Æ2-cen ICAM4 NM_ ICAM-4 LW glycoprotein (IgSF) 17 CH/RG 6p21Æ3 C4A NM_ Complement factor 4A C4B NM_ Complement factor 4B I CD242 3 Bailly et al (1994) S S n.a Yu et al (1986), Yu (1991) Hh 18 H 19q13Æ3 FUT1 NM_ a2fuct II n.a. 365 CD173 1 Kelly et al (1994) Kx 19 XK Xp21Æ1 XK NM_ Xk glycoprotein M Ho et al (1994) Gerbich 20 GE 2q14-q21 GYPC NM_ GPC I CD236R 7 Colin et al (1986) GPD I CD236 Cromer 21 CROM 1q32 DAF NM_ DAF GPI CD55 12 Caras et al (1987), Medof et al (1987) Knops 22 KN 1q32 CR1 NM_ CR1 I CD35 8 Wong et al (1989) Indian 23 IN 11p13 CD44 NM_ Hermes antigen I CD44 2 Goldstein and Butcher (1990), Harn et al (1991) 760 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

4 Table 1. (continued) ISBT Name ISBT number ISBT system symbol Chromosome location ISGN gene symbol Genbank account number* Gene product Protein type Apparent mass (kda) Number of amino acidsà CD number Number of antigens Cloning reference Ok 24 OK 19p13Æ3 BSG NM_ Neurothelin, basigin I CD147 1 Guo et al (1998) Raph 25 RAPH 11p15Æ5 MER2 NM_ MER2 M CD151 1 Hasegawa et al (1996) JMH 26 JMH 15q22Æ3-q23 SEMA7A NM_ H-Sema-L GPI CD108 1 Yamada et al (1999) I 27 I 6p24 GCNT2 NM_ b6glcnact II n.a Bierhuizen et al (1993) Globoside 28 GLOB 3q25 B3GALT3** NM_ b3galnact1, P synthase II n.a Amado et al (1998) GIL 29 GIL 9p13 AQP3 NM_ Aquaporin-3 M Inase et al (1995) *Several different GenBank entries may exist for each system; n.a., not applicable. Most accession numbers given were retrieved from in July Type I and II are single membrane pass molecules with their amino- or carboxyterminals outside the cell (inside the Golgi for glycosyltransferases) respectively. M-n is a multimembrane pass molecule that traverses the membrane n times; GPI is a molecule anchored to the RBC membrane via a glycosylphosphatidylinositol link; S is a molecule that is found in its soluble form in plasma but has been adsorbed to the RBC membrane and covalently bound to lysine residues. àin some instances the number of amino acids given may vary between different variants/forms of the molecule. Whilst the primary gene product is the glycosyltransferase given, the blood group antigens are carried by carbohydrate structures on glycoproteins and/or glycolipids. The size given corresponds to the full length of C4. Observe that the C4d fragment only is adsorbed and bound to the RBC and carries the CH/RG antigens. **Current gene name based on the erroneous assignment of the gene product as a b3-galactosyltransferase. ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

5 Table II. Single nucleotide polymorphisms (SNPs) associated with selected antigens in some important blood group systems. System symbol Antigen 1 Critical SNP (amino acid change) Antigen 2 Reference ABO A C796A, G803C* (Leu266Met, Gly168Ala) B Yamamoto et al (1990a) MNS M C59T, G71A, T72G (Ser1Leu, Gly5Glu) N Siebert and Fukuda (1986) S T143C (Met29Thr) s Siebert and Fukuda (1987) RH C T307C* (Ser103Pro) c Mouro et al (1993), Simsek et al (1994) E C676G (Pro226Ala) e Mouro et al (1993), Simsek et al (1994) LU Lu a A252G (His77Arg) Lu b El Nemer et al (1997), Parsons et al (1997) Au a A1637G (Thr539Ala) Au b Parsons et al (1997) KEL K1 T698C (Met193Thr) K2 Lee et al (1995) Kp a T961C (Trp281Arg) Kp b Lee et al (1996) FY Fy a G125A (Gly42Asp) Fy b Chaudhuri et al (1995), Iwamoto et al (1995), Mallinson et al (1995), Tournamille et al (1995b) JK Jk a G838A Asp280Asn Jk b Olives et al (1997) DI Di a C2561T (Leu854Pro) Di b Bruce et al (1994) Wr a G1972A Glu658Lys Wr b Bruce et al (1995) YT Yt a C1057A (His353Asn) Yt b Bartels et al (1993) SC Sc1 G169A (Gly57Arg) Sc2 Wagner et al (2003) DO Do a A793G (Asn265Asp) Do b Gubin et al (2000) CO Co a C134T (Ala45Val) Co b Smith et al (1994) LW LW a A308G (Gln70Arg) LW b Hermand et al (1995) CROM Tc a G155T (Arg18Leu) Tc b Lublin et al (2000) IN In a C252G (Pro46Arg) In b Telen et al (1996) *Additional missense mutations that differ between these alleles can occur. Another missense mutation at this position encodes a blood group antigen of very low prevalence that is antithetical to antigen 1 and antigen 2. ment of these antigens to a specific blood group system. However, there are exceptions in both cases. For example, the gene responsible for expression of P1 antigen in the P blood group system has not been identified. Despite this, it has been acknowledged as a system of its own since it was declared independent of all other blood group systems. The opposite is true for the P k blood group antigen, currently residing in collection 209. The locus responsible for P k blood group antigen expression was unambiguously shown to be a 4-agalactosyltransferase gene (A4GALT) on the long arm of chromosome 22 (Steffensen et al, 2000). However, since its relation to the P blood group system (also coded for by a locus on the long arm of chromosome 22 according to family studies) is unclear, it remains in the GLOB collection until this issue has been resolved. Molecular mechanisms that generate blood group diversity Diversity in the human genome arises through a number of different mechanisms (Table III) but the most common is the SNP. SNPs are predicted to account for much of the diversity observed between subjects of the same or even closely related species. The 1Æ42 million SNPs originally reported in a genomic map of human genetic variation was just a first hint of the true number of SNPs (Sachidanandam et al, 2001). Since the initial sequencing and mapping of the human genome, the number of SNPs reported has grown exponentially and over 2 million SNPs have been identified and validated ( nih.gov/snp/index.html). SNPs in exon sequences The SNPs can be silent or affect the translated gene product, either as missense mutations, or non-sense mutations. Single amino acid substitutions resulting from missense mutations in exon sequences are common. Accordingly, it is not surprising that it has been estimated that two thirds of all blood group antigens are defined by missense SNPs in blood group genes (Reid & Yazdanbakhsh, 1998). SNPs associated with some important pairs of antithetical antigens are listed in Table II. Non-sense SNPs are those that cause an immediate (and premature) stop codon e.g., a T>A mutation in the FY gene occurring at different points in three unrelated people of the Fy(a b ) phenotype resulted in the substitution of tryptophan by a premature stop codon (Rios et al, 2000a). The mutations at nucleotide 287, 407 or 408 demonstrate the effect of premature stop codons on protein synthesis, since there was no detectable Duffy protein present on the RBCs. SNPs cannot only alter the antigen expressed by a certain blood group molecule but also modify the number of copies expressed in the RBC membrane. This is well illustrated by the many RHD alleles in which a SNP results in weakened expression of the D antigen. Indeed, a single nucleotide change can have a profound effect on the amount of D antigen expressed at the RBC surface, reducing the amount by as much 762 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

6 Table III. Molecular mechanisms that generate diversity in blood group genes. Type of change Molecular mechanism Example of gene event Phenotypic consequence Antithetical antigen Missense SNP KEL 698C>T K2fiK1 antigen Novel antigen Missense SNP GYPB161G>A Mit+ Equal crossover between homologous genes GYP(B-A) S s+ w U, Dantu+ DNA conversion between homologous genes RH(D-CE-D) D VI, BARC+ Exon duplication GYPC.Lsa Ls(a+) Reduced amount of Missense SNP ABO646T>A A x expected antigen FY 298C>T Fy x CROM 596C>T Dr(a ) Splice site mutation GYPB intron 5 +5g>t S s U+ w XK intron 2 +5g>a McLeod phenotype: weakened Kell antigens No protein product Non-sense SNP DO 442T>C Gy(a ) Nucleotide deletion RHAG 1086delA Rh null CO 232delG Co(a b ) Mutation in transcription motif FY 46T>C Fy(a b ) Splice site mutation DO intron 1 2a>g Gy(a ) Gene deletion DRHD D DGYPA En(a ) Modifying gene In(LU) Lu(a b ) *SNP, single nucleotide polymorphism; D, deletion. as 100-fold (Wagner et al, 1999, 2000). Another interesting example is the Fy x phenotype, in which a missense mutation that encodes an amino acid change in an intracellular loop of the molecule results in less Duffy glycoprotein on the cell surface (Olsson et al, 1998; Parasol et al, 1998; Tournamille et al, 1998). The SNP that encodes the Kp a polymorphism not only results in the altered antigen specificity but also affects trafficking of the Kell glycoprotein to the RBC surface so that there is a reduced expression of Kell in the cell membrane whilst increased amounts can be found intracellularly (Yazdanbakhsh et al, 1999). Evolutionary pressure from various pathogens is generally thought to be responsible for the generation of genetic variants, the host effects of which determine whether or not they will survive over time. This has been discussed with a main focus on microbial pathogens, e.g. relating to the differences in carbohydrates expressed on cell surfaces (Gagneux & Varki, 1999) but there is also strong evidence for the role of malaria on the genetic variants of some of the integral RBC membrane proteins, such as the Duffy glycoprotein and the RBC anion exchanger (AE1; band 3) (Miller et al, 1976; Bruce & Tanner, 1999). SNPs in introns and regulatory regions The splice sites are crucial for exon fusion when the introns are removed by the splicing machinery of the cell during the maturation of hnrna to mrna. Mutations that affect the invariant GT at +1 and +2 of the 5 -donor splice site or the invariant AG at 1 and 2 of the 3 -receptor splice site will cause skipping of the preceding or succeeding exons respectively. Mutations of the less conserved nucleotides of the splice site recognition sequence can also affect exon processing and cause exon skipping. The Jk null phenotype in Polynesians (Irshaid et al, 2000) and some K 0 phenotypes (Lee et al, 2001) are examples of how this phenomenon can alter the RBC phenotype. Individuals whose RBCs carry null phenotypes, such as the Jk null and K 0 phenotypes, are at risk of immunization by blood transfusion or pregnancy and once immunized, will require the provision of rare blood for any further transfusion therapy. The S s U+ w phenotype in African- Americans also arises from exon-skipping events due either to a mutation in the intron 5 splice site or to mutations in exon 5 that activate a cryptic splice site (Storry et al, 2003). The SNP responsible for the Dr(a ) phenotype in the Cromer blood group system, also creates a cryptic splice site in the DAF gene that is used preferentially (Lublin et al, 1991). The product of the alternative splicing is not found on the RBC surface and consequently, Dr(a ) RBCs express only 40% of normal levels of DAF. The SNPs that occur in the regulatory elements, such as the promoter or enhancer regions of blood group genes, can modify or abolish antigen expression. A well-known example of such modification is the altered tissue distribution of the Duffy blood group antigens commonly found in individuals of African origin. A disruption of the GATA-1 motif in the promoter region of the FY*B gene by a single nucleotide substitution abolishes erythroid expression whilst the molecule is expressed normally in other tissues (Tournamille et al, 1995a). This mutation is a perfect illustration of evolutionary pressure exerted by a pathogen since the Duffy protein is the exclusive receptor on mature RBCs for the malarial parasite, ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

7 Fig 1. Hybrid genes arising from crossover events between ABO genes have been shown to give rise to more or less unexpected phenotypes. In this example, a crossover in intron 6 between an O 1v allele and a B allele to generate a B O 1v hybrid was shown to be one molecular mechanism behind the A x phenotype (Olsson & Chester, 1998). Weak A antigen expression occurs because exon 7 of the O 1v allele encodes A transferase activity that is normally silenced by the presence of 261delG in exon 6. Since exon 6 is derived from the B allele without this deletion in the hybrid, enzyme activity is restored. The corresponding product of the crossover would be expected to encode a non-functional protein since the 261delG mutation is present in exon 6. Indeed, such an allele was reported to be common in Brazilian blacks (Olsson et al, 1997) and has since been found in Blacks of several different geographic origins (unpublished observations). Note that the introns are not drawn to scale. Plasmodium vivax (Miller et al, 1976). The Duffy protein is absent from the RBCs of up to 100% of native West Africans and consequently, these individuals are protected from P. vivax infection. Furthermore, the same evolutionary pressure is proposed to have driven the identical mutation in the GATA-1 sequences of the FY*A allele in a Papua New Guinean population but, interestingly, the mutation is thought to be a much more recent and unlinked event (Zimmerman et al, 1999). Other mechanisms that contribute to blood group diversity While single nucleotide changes can have far-reaching consequences on gene product expression and function, there are also other mechanisms that contribute to diversity. Gene rearrangements due to recombination or gene conversions between homologous genes, such as those encoding the Rh and MNS blood group systems, can affect blood group expression in many different ways (Blumenfeld & Huang, 1997; Avent, 2001). Surprisingly, the same is true for ABO where only a single gene locus results in multiple hybrid alleles, giving rise to unexpected phenotypes (Fig 1) (Olsson & Chester, 2001). Recombination between the two homologous genes in the Rh and MNS blood group systems is common and can lead to many different kinds of phenotype. Examples of exchange between homologous genes in trans are common in the MNS system where hybrids of GPA and GPB are created by unequal crossover or gene conversion events (Fig 2A) (Blumenfeld & Huang, 1997). New antigens arise as a result of the novel amino acid sequences generated by the hybrid genes. The hybrid molecules that carry unusual phenotypes in the Rh blood group system are thought to be generated by crossover between the RHD and RHCE genes in cis (Fig 2B) (Wagner et al, 2001), that may alter or abolish the expression of expected antigens and create novel antigens/phenotypes. For example, the partial D phenotype, D VI type I, results from a RH(D-CE-D) hybrid in which exons 4 and 5 of RHD are replaced by the corresponding exons of an RHcE allele (Avent et al, 1997; Huang, 1997). The hybrid protein expresses a qualitatively and quantitatively altered D antigen. A similar exchange of RHD with exons 4, 5 and 6 of an RHCe allele produces a hybrid protein with a qualitatively identical D antigen, as determined by monoclonal antibody studies; however, more copies of the D antigen are present and these RBCs also express the low incidence antigen, BARC (Mouro et al, 1994; Tippett et al, 1996). Non-sense mutations, such as nucleotide deletions or insertions, often abolish or decrease blood group expression by causing a shift in the open reading frame of the sequence such that the amino acids encoded after the mutation are completely different. For example, in the ABO system, two different single nucleotide deletions in the consensus A 1 sequence have been shown to account for the common O and A 2 blood groups. These are illustrated in Fig 3 (Yamamoto et al, 1990a, 1992). Not surprisingly, the deletion of whole genes or parts of genes can result in loss of blood group antigen expression as exemplified by the following reports concerning the MNS (Huang et al, 1987; Rahuel et al, 1988), RH (Wagner & Flegel, 2000), JK (Irshaid et al, 2002), H (Koda et al, 1997) and GE (Colin et al, 1989) blood group systems. Duplication of genetic material can result in the formation of a novel antigen, for example, the nucleotide sequence created by the exon 3-exon 3 duplication in the GYPC.Ls a variant gene encodes the Ls a antigen (Reid et al, 1994). Conversely, a duplication event may result in the loss of an existing antigen. 764 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

8 Fig 2. Mechanisms that generate hybrid genes in the MNS and Rh blood group systems. (A) DNA exchange between the GYPA and GYPB genes in trans, either by unequal crossover or by gene conversion, creates novel sequences that are recognized as blood group antigens. (B) Crossover of the RH genes in cis creates hybrid genes that have altered expression of expected antigens and may create novel antigens. SMP is an unrelated gene. Genes are illustrated in a 5 3 direction unless noted otherwise. Novel sequences are indicated by the parenthesis. The best example of this is the 37 bp nucleotide duplication that occurs at the intron 3/exon 4 border in the RHD pseudogene (Singleton et al, 2000). The duplication results in an alteration of the reading frame and an eventual premature stop codon so that no RhD protein is found on the RBCs. In the ABO gene, a 43 bp minisatellite motif 4 kbp upstream from exon 1, with the ability to bind the transcription factor CBF/NF-Y has been suggested to govern transcription in an enhancer-like way (Kominato et al, 1997). Allele-related variation in the number of repeats was observed in samples of different ABO genotypes: A 1 and O 2 alleles having one copy only while A 2, B, O 1 and O 1v alleles had four copies (Irshaid et al, 1999). In an experimental model, four repeats (associated with the common A 2, B and O 1 /O 1v alleles) produced approximately 100 times more mrna than a single repeat (found in A 1 and O 2 alleles) (Yu et al, 2000). Blood group genes that control carbohydrate antigens All genes encode proteins but not all blood group genes encode blood-group-carrying proteins. The reason, of course, is that not all blood groups antigens are of protein nature. This apparent anomaly was indeed confusing to the pioneers who had elucidated the biochemical basis of blood groups like ABO, H and Lewis. On one hand, DNA was supposed to code for proteins but, on the other hand, these carbohydrate blood groups were definitely inherited characters. The solution came when Watkins hypothesized that the genes encoded bloodgroup-specific glycosyltransferases (Watkins, 1974). This hypothesis held true, although today researchers still struggle to clarify the genetic heterogeneity underlying variant carbohydrate blood groups. In theory, any mutation that changes the enzymatic properties of the primary gene product including ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

9 Fig 3. A single nucleotide deletion in the coding region of a gene can alter the open reading frame. For example, in the ABO blood group system, the deletion of 261G in the consensus sequence (A 1 allele) results in a frameshift and a subsequent introduction of a premature stop codon (O 1 allele). The truncated protein that is encoded is inactive. Conversely, the A 2 allele results from the deletion of 1061C, and instead, the open reading frame is elongated. The glycosyltransferase encoded by this gene consists of 21 additional amino acids and is less efficient, as determined by the presence of fewer A antigens on the RBCs of group A 2 people. In addition, it appears to be unable to synthesize the A 1 antigen. Asterisks (*) represent stop codons. The grey shading indicates nucleotides that are transcribed normally; the white boxes indicate a nucleotide sequence that is not transcribedas a result of the nucleotide deletion at position 261; the hatched box represents the additional nucleotide sequence that is transcribed as a result of the nucleotide deletion at activity, substrate and acceptor preference may cause, e.g. a weak A or B subgroup (Chester & Olsson, 2001). The null phenotypes O, p, or Bombay result from the inheritance of two inactive alleles of the respective glycosyltransferase gene. In all three genes, inactivating mutations have been found throughout the coding sequence. For ABO, however, a singlenucleotide deletion is the predominant cause of the blood group O phenotype whilst other causes are infrequent or rare. Currently, the number of alleles at the ABO locus approaches a hundred and reaches a degree of complexity due to point mutations and hybrid allele formation that is only matched by the RH and MNS loci. Heterogeneity of null phenotypes By comparison with the ABO locus, most of the protein-based systems are relatively simple and a polymorphism defined at the genetic level can almost always be correlated with the expression of a certain blood group antigen. However, in genomic DNA-based analysis, the interpretation of results can be confounded by the existence of null phenotypes. Clearly, any mutation in the gene that results in the failure of the antigen-carrying molecule to be expressed at the RBC surface will, in fact, be a null mutation. Because some of these mutations occur relatively commonly, it is important to include their detection together with assays for common phenotype prediction in order to avoid false positive results. For example, it is necessary to test for the GATA-1 mutation in the FY gene described above, when determining Fy a and Fy b antigen status but not for the other very rare mutations that result in the Fy(a b ) phenotype. Similarly, in the Nordic population, it should be considered to include detection of the 871T>C mutation in the JK gene that is the basis for the Jk(a b ) phenotype in the Finnish people (Irshaid et al, 2000). The heterogeneity of the molecular bases of these phenotypes is a major problem for all DNA-based prediction of blood groups. Most null blood group phenotypes are the result of molecular changes in the gene that encodes the carrier molecule. However, there are important interactions between proteins at the cell surface and with the cytoskeleton and therefore mutations that change the expression of an interactive protein can affect the proteins around it. Mutations in RHAG that stop the expression of the Rh-associated glycoprotein (RhAG) also prevent the expression of the RhD and RhCE proteins the so-called regulator type of Rh null phenotype (reviewed in Daniels, 2002). Similarly, mutations leading to the absence of the XK protein in the red cell membrane results in the weakened expression of Kell blood group antigens (Danek et al, 2001; Russo et al, 2002). Future perspectives Today, almost all of the genes underlying expression of the human blood group systems have been cloned and the polymorphisms responsible for the phenotypes encountered in different individuals and populations clarified. A challenge that remains for the future is to investigate and understand the genetics of antigens in the blood group collections and series since our experience tells us that they are likely to be carried on functional molecules. The increasing knowledge of the genes encoding blood group antigens has relevance in the clinical laboratory. Genomic typing assays for fetal RBC phenotype prediction to determine the risk to a foetus of haemolytic disease of the newborn are now standard of care. Much of this testing is performed on DNA isolated from amniotic fluid; however, in the last few years, more sensitive quantitative PCR techniques have permitted the identification of fetal RHD alleles from DNA isolated from maternal plasma (Lo et al, 1998; Lo, 2001; 766 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126,

10 van der Schoot et al, 2003). Although there are some current limitations to the technique, the advantages of this noninvasive method are obvious. Other applications of blood group genotyping have included testing samples from multiply transfused patients that are either immunized, or at risk of being immunized, to one or more blood group antigens (Reid et al, 2000). Patient groups, such as those with Sickle Cell Disease or other transfusion-dependent haemoglobinopathies, can benefit from better antigen-matched blood (Reed & Vichinsky, 2001). Genotyping is also useful in other serological situations where the RBC phenotype cannot be accurately determined (Rios et al, 2000b). The potential for testing blood donor samples on a large scale is clear to all in the field, but current techniques are both laborious and expensive. However, automation of SNP detection, as a faster and easier way to type blood donors, is a much-discussed issue and there are several techniques being evaluated. Lastly, the knowledge gained from the identification of blood group genes leads to a better understanding of the relationship between blood group differences and subtle functional differences in the molecules that carry the antigens. The molecular genetics of blood groups may also help us to better understand the functionality of the RBC at large, e.g. moderation of the intracellular environment for invading parasites, plasticity of the RBC membrane in the circulation during stress, or the role of the circulating RBC in haemostasis. References Amado, M., Almeida, R., Carneiro, F., Levery, S.B., Holmes, E.H., Nomoto, M., Hollingsworth, M.A., Hassan, H., Schwientek, T., Nielsen, P.A., Bennett, E.P. & Clausen, H. (1998) A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose: beta-n-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family. 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