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Various techniques of genotyping the MNSs blood group have been described, but none of them enables the complete detection of all MNS antigens.
Blood samples were obtained from blood donors. Primers were created using the published DNA sequences for glycophorins A and B. Genotyping was performed using polymerase chain reaction sequence-specific primers (PCR-SSP).
A total of seven primers were found to specifically amplify the most common MNS antigens. The use of these primers has enabled us to correctly genotype all blood samples tested so far (n=116).
Specifically created primers enable genotyping of the MNS antigens in a single PCR-SSP run. The method is reliable, easy to perform, and can be used in routine practice.
The MNS blood group system includes at least 40 antigens, the most important of which are M and N on glycophorin A (GPA), and S and s on glycophorin B (GPB)1. The remaining antigens are very rare. The MNS blood groups can be typed by serological methods, allele-specific polymerase chain reaction (PCR)2–5, reverse dot blot hybridization6, PCR-restriction fragment length polymorphism or inverse PCR7, reverse transcriptase-PCR8 or single strand conformation polymorphism2. However, none of the previously described genotyping methods allows detection of all MNS antigens in one step, not least because the creation of MNS-specific primers is rather difficult. The genes for glycophorins A, B and E (GPA, GPB and GPE, respectively) contain a highly homologous area spanning the first four exons9–15.
The differences between GPAM and GPAN are reflected by an amino acid exchange at positions 1 (Ser to Leu) and 5 (Gly to Glu) in the distal part of the mature protein. GPBS and GPBs are differentiated by a single amino acid exchange from Met to Thr at position 29. These amino acid exchanges are produced by base exchanges C2T, G14A and T15G in exon 2 for M and N, and by a single base exchange C1822T for S and s1,10. In addition, there is only one base exchange in intron 1 (G-28T)10, and only two relevant base exchanges (G377A and C395A) occur in intron 2.
The tremendous amount of antigens included in the MN system is an additional problem. Some of the rare antigens were found to be produced by a single base mutation involving Vr (GPAC197A) and Mta (GPAC230T)16. This kind of allele can only be typed using specially assigned primers. The other rare antigens result from exchanges between different glycophorin genes in various parts of the sequence17. As a result, the final antigen assignment can be very difficult because rearrangement may abolish the sequence of one primer, thereby producing false homozygosity.
Based on the published sequences for glycophorins A, B and E, we created new primers that allow complete detection of all common MNS antigens.
Ten primers (Table I) were designed on the basis of published sequences for glycophorins A, B and E18. The PCR mix included: 11.3 mM Tris-HCl, 56.4 mM KCl, 1.7 mM MgCl2, 0.9 mM dNTP-mix (0.29 μmol per dNTP), 0.001% gelatin, 0.28% glycerol, 0.01% cresol-red, 0.5 U Ampli-Taq® (Roche Molecular Systems, New Jersey, USA), 48 ng genomic DNA, and 25 pmol of the 5′ and 3′ primers. The final volume was 10 μL. The cycling conditions were: 2 min at 94°C, 10 cycles of 10 sec at 94°C and 1 min at 65°C, and 20 cycles of 10 sec at 94°C, 50 sec at 61°C and 30 sec at 72°C. The products were run on an agarose gel for 20 min at a rate of 13 V per cm and stained with ethidium bromide.
DNA was isolated from 10 mL EDTA peripheral blood samples using a standard salting out technique (Super Quik-Gene from Immucor, Rödermarck, Germany). DNA was adjusted photometrically to a concentration of 100 ng/μL after solubilization in distilled water.
The created primers were used to genotype a total of 116 samples from healthy blood donors of Caucasian origin with known serological MNS antigens. The following phenotypes were tested: M+N+S+s+ (n=29), M+N+S-s+ (n=28), M+N-S+s+ (n=19), M-N+S-s+ (n=16), M+N-S-s+ (n=12), M-N+S+s+ (N=4), M+N-S+s+ (n=4), M+N+S+s- (n=3) and M-N+S+s- (n=1).
Eight primer pairs for MNS typing were used (Table II). Four primer pairs for M and two primer pairs for N were assigned to obtain information about the GPA gene. All primer pairs were used under the same PCR conditions and yielded stable amplification results.
The primers in intron 1 (Mf1, Nf1) produced only faint reactions with either of the MN-reverse primers. Moreover, primer Mf1 did not react with an allele of M (MT)2,7,19. Nf1 and Nr2 are present on glycophorins A, B and E and do not allow specific MNS genotyping. The genomic sequence for GPA shows variable base mismatches inside the Mr1 and Nr1 sequences. These two primers, therefore, yielded variable amplification results in different antigen-positive donors and could not be used for testing purposes. The primer pair Mf3–Mr2 resulted in only very faint amplification (Figure 1).
The reaction patterns of MNS typing for the different serological combinations are shown in Figure 2. These results were found to be in 100% concordance with those of serological typing. Reaction failure or inconclusive results have not been observed in any of the non-variant samples tested so far.
The small number of relevant base exchanges in exon 2, intron 1 and intron 2 of GPA and the pronounced homology between the various glycophorin genes pose a special problem when creating primers for genotyping the M and N blood groups10. The fact that one allele of GPAM (MT) has thymine at position −28 poses another difficulty. When located at this position, this rare allele cannot be differentiated from N2,19. This position is, therefore, unsuitable for creating specific primers. Ideally, one primer should be specific for antigen polymorphism while the counter primer is specific for the glycophorin gene itself. We found this strategy to be best suited for MNS genotyping. The primers we selected provide redundant information on different locations between introns 1 and 2 of the GPA gene. Of these various primers, four primer pairs were selected for M genotyping and two for N genotyping. Although the exchanged sequence of most rearranged genes starts behind the tested area in introns 1–2, some hybrid antigens can be defined by this method. For example, the hybrid molecule GP.Vw (rearrangement pattern A-B-A, formerly Mi I) was only detected by primer mix 5. In this case, the amplification failure of some mixes in our typing kit suggests the possible presence of a rare GPA allele. The results for all MN antigen complexes tested were found to be in concordance with the serological results. Our primers may help to resolve the problem of defining specific primers for S and s20. The results for all S and/or s-positive combinations tested were in concordance with the serological results. We conclude that our primers are currently best suited for genotyping of MNS antigens in laboratories performing only a simple PCR-technique.