STATE OF THE ART
Miltenberger phenotypes are glycophorin variants: a review
Christine Lomas-Francis, Laboratory of Immunohematology and Genomics, New York Blood Center, 45-01 Vernon Boulevard, Long Island City, NY 11101, USA
Antigens of the MNS blood group system are expressed in the red cell (RBC) membrane on two sialic acid-rich glycoproteins, glycophorin A (GPA) and glycophorin B (GPB) or on hybrid molecules composed of portions of GPA and GPB. GPA and GPB are encoded, respectively, by the homologous genes GYP*A and GYP*B. Another homologous gene, GYP*E, participates in gene rearrangements but may not encode a RBC membrane component. The MNS system is highly polymorphic and, at the time of writing consists of 46 distinct antigens [1,2]. Many are antigens of low-prevalence and a number of these were, for many years, grouped together in the Miltenberger series or subsystem. This subsystem was introduced by Cleghorn in 1966  in an effort to classify several rare phenotypes that appeared to be related to each other through reactivity with the serum of Mrs. Miltenberger and through overlapping expression of certain low-prevalence antigens (namely, Mia, Vw, Mur, Hil). These phenotypes were initially associated with the MNS blood group system by the observation that Verweyst (Vw) was inherited with MNS. Cleghorn collated and extended the studies of several laboratories with various sera and originally defined four classes (Mi.I to Mi.IV) on the basis of their different reactions with four type sera called Vw, Miltenberger (Mia), Murrell (Mur) and Hill (Hil). Later, samples were found that did not react with the Miltenberger serum yet reacted with one or more of the other three original type sera. For example, RBCs with the Mi.V phenotype reacted with anti-Hil, but not with the Miltenberger serum whereas the Mi.III phenotype reacted with both. From the outset, the serological testing was complicated because few of the reactive sera were monospecific. By 1992, the Miltenberger subsystem had expanded to 11 classes . Although a sequence of amino acids representing the elusive Mia determinant had been proposed , there was increasing doubt as to the existence of the Mia antigen as, at that time, a separate anti-Mia had not been found nor could it be isolated from the serum of Mrs. Miltenberger. Almost a decade later, believers in the existence of the Mia antigen were vindicated by the production of monoclonal anti-Mia [5,6].
As the complexity of the Miltenberger subsystem increased, its further expansion no longer seemed feasible or relevant; it was declared obsolete and a new terminology was introduced. The terminology for the serologically defined phenotypes is based on ‘GP’ for glycophorin and ‘GYP ’ for the gene, with the addition of the abbreviated name of the person in whom the variant was first described, for example, Mi.I became GP.Vw (encoded by GYP*Vw), Mi.II became GP.Hut (encoded by GYP*Hut) and Mi.III became GP.Mur (encoded by GYP*Mur) [7,8]. Table 1 shows the GP classification and the associated low-prevalence antigens.
Table 1. Glycophorin (GP) classification and associated low-prevalence antigens of the obsolete Miltenberger subsystem
Investigation of MNS variants at the level of the gene and the protein showed that the phenotypes, previously placed in the Miltenberger subsystem, are expressed by hybrid GP molecules: GP(A–B), GP(A–B–A) and GP(B–A–B). Other hybrids, for example, GP(B–A), and GP(A–A), have been described  but this review will focus on the serologic, molecular and genetic information of the GP variants that were previously referred to as Miltenberger phenotypes.
The glycophorins and the genes that encode them
GPA and GPB are single pass sialic acid-rich GPs that are heavily glycosylated with numerous O-glycans and GPA also carries an asparagine-linked glycan (N-glycan). GPA is the most abundant sialoglycoprotein in the RBC membrane (1 × 106 copies per RBC). It consists of 131 amino acids, organized into three domains: an extracellular N-terminal domain of 72 amino acids, a hydrophobic membrane-spanning domain of 23 amino acids and a C-terminal cytoplasmic domain of 36 amino acids. GPB (2 × 105 copies per RBC) is similar to GPA, but consists of 72 amino acids also organized into three domains: an extracellular N-terminal domain of 44 amino acids, a hydrophobic membrane-spanning domain of 20 amino acids and a short C-terminal cytoplasmic domain of 8 amino acids. Both GYP*A and GYP*B encode a leader sequence of 19 amino acids that is cleaved in the mature protein. Current convention is to number the amino acids in a protein starting with #1 at the initiation codon of Met (Methionine). This means a change from the numbering used in most previous publications. In this review, the current amino acid number will be used but the historical number will be given in parentheses.
The homologous genes, GYP*A and GYP*B, are located on the long arm of chromosome 4 (4q31) . A third homologous gene, GYP*E, which may not encode a RBC product, is adjacent to GYP*B and participates in gene rearrangements resulting in hybrid alleles . GYP*A, GYP*B and GYP*E share more than 95% sequence identity. The genes reside in an approximately 350-kb gene cluster (5′-GYP*A–GYP*B–GYP*E-3′). The intron–exon structure of the genes is similar: GYP*A has seven exons, GYP*B has six exons (of which exon 3 is a pseudoexon or non-coding exon) and GYP*E has six exons (of which exons 3 and 4 are pseudoexons). For each of the GYP genes, exon 1 and part of the 5′ end of exon 2 encode the leader sequence for the corresponding GP, exons 2–4 encode the extracellular domains, exon 5 encodes the transmembrane domains of each GP and exon 6 and part of the 5′ end of exon 7 of GYP*A encode the cytoplasmic domains. In GYP*A, exon 3 is fully functional and expressed; however, exon 3 in GYP*B is usually not expressed because of the presence of a point mutation in the acceptor splice site. At meiosis, when gene rearrangements can occur, the defective splice site may be ‘repaired’ leading to expression of novel GPs . If GYP*E encodes a membrane-bound product (this is still controversial), it would be a short sialoglycoprotein with the M antigen at its amino-terminus. The sequence homology between the GP genes and organization along the chromosome is thought to be responsible for the relatively frequent occurrence of recombination, unequal cross-over and gene conversion events among the three GYP genes.
Molecular basis of variant phenotypes
Variant phenotypes may occur as a consequence of a single amino acid substitution, cross-over, gene conversion or gene deletion (Table 2). Recombination hotspots have been identified and are mainly clustered in a 2-kb stretch of genomic sequence that encompasses exons 2–4; these exons encode the extracellular domains of GPA and GPB. The reasons for the rearrangement are unknown but they have resulted in many hybrid genes and consequently in the antigenic diversity of the MNS system (Table 3). A detailed description of the various mechanisms is beyond the scope of this review; more information may be found in numerous reviews including those by Huang et al., Reid and Palacajornsuk [11–13].
Table 2. Molecular mechanisms that encode variant MNS phenotypes and the associated antigens
|Single nucleotide substitution||GPA: Vr, Mta, Ria, Nya, Or, ERIK, Osa, ENEP/HAG, ENAV/MARS, ENEV, MNTD |
GPB: S/s, MV, sD, Mit
|Two or more nucleotide substitution||GPA: M/N |
|Unequal crossing over||Sta, Dantu, Hil, TSEN, MINY, SAT|
|Gene conversion||He, Mia, Mc, Vw/Hut/ENEH, Mur, Mg, Me, Sta, Hil, Hop, Nob, DANE, MINY, MUT, ENDA|
Table 3. MNS system hybrid alleles, encoded glycophorins (GPs), phenotypes and associated low-prevalence antigens
|GYP*(B–A–B)||GP(B–A–B)||GP.Mur||Mia, Mur, Hil, MINY, MUT|
|GP.Bun||Mia, Mur, Hil, Hop, MINY, MUT|
|GP.HF||Mia, Hil, MINY, MUT|
|GP.Hop||Mia, Mur, Hop, TSEN, MINY, MUT|
|GP(A–B)||GP.He (P2, GL)||He|
|GP.Hut||Mia, Hut, MUT|
|GYP*A 179G>A||GPA||GP.EBH||ERIK encoded by one transcript|
|GP(A–A)||GP.EBH||Sta encoded by a second transcript|
MNS antigens may arise from single amino acid changes in GPA or GPB, or from novel amino acid sequences formed at the junction of GPA to GPB, or GPB to GPA in a number of different hybrid GP molecules or from the expression of amino acids encoded by the pseudoexon of GYP *B (reviewed in Reid and Lomas-Francis  and by Huang and Blumenfeld ). These variant GPs may be detected by altered expression of M, N, S or s antigens, an unexpected sensitivity or resistance of these antigens to treatment of RBCs by proteolytic enzymes, the absence of a high-prevalence antigen, the presence of a low-prevalence antigen or a change in mobility observed during analysis by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis or Western immunoblotting (reviewed in Reid ).
Table 1 shows the GP classification, the type of hybrid GP molecule and the low-prevalence antigens expressed by the GP variants that constituted the now obsolete Miltenberger subsystem. These variants can be placed in the categories described next.
Glycophorin A–B hybrids: GP(A–B)
GP.Hil and GP.JL are each encoded by a GYP*(A–B) hybrid gene. The GYP*A to GYP*B junction in GYP*Hil is located at the 5′ end of intron 3 of GYP*A, whereas in GYP*JL the junction occurs at the 3′ end of intron 3 and includes seven nucleotides of exon 4 of GYP*B. The GP.Hil phenotype travels with either Ms or Ns, has elevated expression of s but weakened M or N expression, and expresses Hil and MINY antigens [14,15]. GP.JL expresses an altered S, a weak M, TSEN and MINY antigens. The S antigen of TSEN+ RBCs does not react with all anti-S, including anti-S produced from the MS94 monoclonal cell line. GP.JL has been found in Europeans, people from southern China and in Hispanics . Most examples of the GP.Hil phenotype have been found in people with European ancestry; GP.Hil was found with a prevalence of 1 in 2,000 in one survey of Swiss blood donors  and was found in one Taiwanese. A Spanish-American woman, who was homozygous for GYP*Hil, and two people who were GYP*Hil/Mk have been reported .
Glycophorin A–B–A hybrids: GP(A–B–A)
GP.Vw, GP.Hut, GP.Nob, GP.Joh and GP.Dane are each encoded by a GYP*(A–B–A) hybrid. In these hybrid genes, small inserts (from 1 to 16 bp) of different short portions of the pseudoexon of GYP*B replace the same number of nucleotides in exon 3 of GYP*A and a part of the pseudoexon is translated.
The insert encoding GP.Vw and GP.Hut results in an amino acid polymorphism at position 47 (28); the threonine present in GPA is changed to methionine in the case of GP.Vw and for GP.Hut to lysine. GP.Vw and GP.Hut phenotype RBCs are recognized by anti-Vw and anti-Hut, respectively. The allele responsible for GP.Vw travels mostly with Ns, occasionally with NS, and was found only once with MS whereas GP.Hut travels equally with MS or Ns. The highest prevalence of the GP.Vw phenotype, 1·43%, was found in southeastern Switzerland .
RBCs with GP.Nob express the Nob antigen. GP.Nob differs from GPA at amino acid positions 68 (49) and 71 (52); the arginine at position 68 in GPA is substituted by threonine and the tyrosine at position 71 is replaced by serine because of 10 nucleotides (67–76) in exon 3 of GYP*A that have been replaced by the corresponding sequence of the GYP*B pseudoexon [20,21]. The GP.Nob phenotype has been only found in white donors; tests on 4929 random group O blood donors in Bristol, England, found a prevalence of 0·06% . GP.Nob is associated with Ms and MS.
GP.Joh closely resembles GP.Nob but expresses Hop as well as Nob antigen. The altered GPA of GP.Joh differs from GP.Nob by having only the arginine to threonine change at amino acid position 68 (49) . The prevalence of GP.Joh is unknown. The GYP*Joh travelled with Ns in the families of the two known probands.
Two different hybrid GYP*(A–B–A) genes encode the GP.Dane phenotype that is characterized by expression of the low-prevalence antigens DANE and Mur and a trypsin-resistant M antigen; at the RBC level, the products of the two alleles are indistinguishable. GP.Dane was characterized by a short sequence of amino acids encoded by exon 3 (the pseudoexon) of GYP*B (54Pro-Ala-His-Thr-Ala-Asn59) and in the first proband by an additional substitution of Ile65 (46) to Asn64 (45) caused by an adenyl nucleotide change. GP.Dane has a prevalence of 0·43% in Danes. In the four Danish probands, GYP*Dane was inherited with MS . In the more recently identified American woman with English ancestry, GYP*Dane was inherited with Ms and was in trans to an Mk allele; furthermore, her RBCs lacked the high-prevalence antigen ENDA .
Glycophorin B–A–B hybrids: GP(B–A–B)
GP.Mur, GP.Hop, GP.Bun and GP.HF are each encoded by a GYP*(B–A–B) hybrid. In these hybrids, the normally silent GYP*B pseudoexon 3 is expressed. GP.Mur and GP.Bun are similar phenotypes: the RBCs are Mur+, Hil+, MINY+ and MUT+ but GP.Bun expresses Hop whereas GP.Mur RBCs are Hop− (Table 1). GP.Mur and GP.Bun are always inherited with the s antigen; for GP.Bun this is generally with Ms. In people of European ancestry, GP.Mur may be inherited with either Ms or Ns, the latter being more frequent. In Thais and Chinese, GP.Mur usually travels with Ms. RBCs with the GP.Mur or GP.Bun phenotype have elevated expression of the trypsin-resistant N antigen (‘N’) carried on GPB. The s antigen associated with GP.Mur is unusual, it may appear enhanced in serologic expression, yet is not detected by some potent anti-s. That it must also differ qualitatively, and encode a partial antigen was demonstrated by an s+ woman with the GP.Mur phenotype who made alloanti-s (cited in Daniels ). A patient with the GP.Mur/GP.Mur phenotype and U+ RBCs made an apparent anti-U that defines an epitope incorporating an amino acid sequence encoded by GYP*B at the exon 2–4 junction, which is not expressed on GP.Mur, GP.Hil or S-s-U- cells . Both GP.Mur and GP.Bun are encoded by a GYP*Bs allele but differ in the length of the GYP*B pseudoexon insert (55 bp for GP.Mur and 131 bp for GP.Bun) . This segment comprises a portion of both exon 3 and intron 3, and the rearrangement results in the expression of a normally unexpressed GYP*B pseudoexon sequence. The GYP*Bun allele differs from the GYP*Mur allele by only one nucleotide in the coding sequence. This is predicted to result in arginine (GP.Mur) or threonine (GP.Bun) at position 57 (48). GP.Mur and GP.Bun are rare in Caucasians. The GP.Mur variant is relatively common in southeast Asia with a prevalence of 9·6% in Thais, 5% in most Chinese populations, 7·3% in the general Taiwanese population but with a greater prevalence (up to 88%) in some indigenous Taiwanese tribes [28,29]. Presence of GP.Mur may provide resistance to Plasmodium falciparum . The presence of GP.Mur in the RBC membrane can up-regulate the amount of band 3 on the RBC surface thereby making the RBC more resistant to osmotic stress .
GP.Hop, which expresses TSEN but not Hil, is identical to GP.Bun except that the allele responsible for GP.Hop encodes S, whereas the allele for GP.Bun encodes the s antigen. Only two GP.Hop probands, both Caucasian, have been described. The S antigen expressed by GP.Hop is unusual and similar in reactivity to the S expressed on GP.JL (see before). GP.HF is characterized by the presence of M, elevated ‘N’ and an unusually strong s antigen as well as by its reactivity with anti-Hil, -MINY and -MUT. This GP hybrid is similar to GP.Mur and GP.Bun. In GP.HF, a 98 bp insert from exon 3 of GYP*A creates a GYP*(B–A–B) hybrid, which encodes a peptide differing from GP.Mur by five amino acid residues and from GP.Bun by six amino acid residues [32,33].
Investigation and identification of glycophorin variants
RBCs with altered GPs may be detected in a variety of ways. The M, N, S or s antigens are expressed more strongly or more weakly than controls, and are unexpectedly sensitive or resistant to treatment by different enzymes; there is a loss of a high-prevalence antigen, or appearance of a low-prevalence antigen. In addition to testing with antibodies to low- and high-prevalence MNS antigens, insight can be gained by the use of relatively simple serological tools such as tests with enzyme-treated RBCs. On intact RBCs, GPA is susceptible to cleavage by trypsin at amino acid residues 50 (31) and 58 (39) but is resistant to α-chymotrypsin cleavage whereas GPB is resistant to trypsin cleavage but sensitive to α-chymotrypsin at amino acid residue 51 (32) . Many GP variants have altered susceptibility to proteolytic enzymes; for example, the M antigen on GP.Dane is trypsin resistant; the Mia antigen has different enzyme susceptibilities when expressed on GP.Vw and GP.Hut when compared with the Mia antigen expressed on GP.Mur and GP.Hop. Testing of RBCs with supernatant fluids containing monoclonal antibodies to various portions of GPA or GPB can deduce the portions of GPA and/or GPB expressed. Tests with lectins detect altered glycosylation of GP variants. Analysis by SDS-polyacrylamide gel electrophoresis will reveal altered mobility caused by either a new GP molecule or a change in glycosylation . Immunoblotting  and polymerase chain reaction (PCR)-based techniques such as PCR–restriction fragment length polymorphism, PCR sequence-specific primer assays [29,35] and cloning or sequencing of relevant GYP exons  have been used for detection and identification of GP variants. Inhibition with synthetic peptides or tryptic digests of GPA or GPB, or epitope (Pepscan) analysis can reveal the antigenic determinant detected by a particular antibody [4,5]. Haemagglutination techniques can focus an investigation on the most relevant part of the gene or molecule.
Antibodies detecting high- or low-prevalence MNS antigens and their clinical relevance
Antibodies to many low-prevalence MNS antigens are clinically relevant: for example, anti-Mia, -Vw, -Hil, -Hut and -Mur have caused haemolytic disease of the foetus and newborn (HDFN) and in some cases the HDFN was severe. These antibodies also have been implicated in (delayed) haemolytic transfusion reactions [30,36–40]. However, once identified, providing blood negative for the corresponding antigen should not be difficult. Alloantibodies produced by individuals lacking high-prevalence antigens on GPA are broadly defined as anti-Ena; they can be further categorized as anti-EnaTS, -EnaFS (recognizing, respectively, trypsin or ficin-sensitve determinants) or as anti-EnaFR when the determinant is ficin resistant. Anti-Ena are made by people with altered GPA such as those found on ENKT−, ENEP−, ENEH−, ENAV− or ENDA− RBCs. Expression of the high-prevalence antigen Wrb requires the presence of amino acids 75–99 (56–79) of GPA. These amino acids are absent in GP.Mur, GP.Bun, GP.HF, GP.Hop, GP.Hil and GP.JL allowing those who are homozygous or hemizygous for the gene encoding these variants to make anti-Wrb or anti-Ena/Wrb. Such antibodies have caused transfusion reactions and finding suitable blood is challenging. In ENEP−, ENAV− or ENEV− RBCs, where the alteration is a single amino acid change in the portion of GPA that interacts with band 3 the expression of Wrb is altered [26,41]. In the only known ENEV− proband, anti-ENEV caused a delayed transfusion reaction on several occasions .
The application of hemagglutination techniques and molecular analysis now allows the elucidation of the nature of even complex GP variants. The remaining challenge associated with Miltenberger phenotypes is to overcome the confusing, frequently incorrect terminology in common usage. Readers of this review will understand that anti-Mi.III or the Mi.III antigen does not exist and that a reaction with GP.Mur phenotype RBCs does not equate to the presence of (only) anti-Mia.
No potential conflict of interest to declare.