PCR-based genotyping of MNSs blood group: Subtyping of M allele to MG and MT

Abstract

SummaryPCR-based genotyping of MNSs blood group system was investigated in combination with restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP) and allele-specific PCR amplification (ASPA) techniques. M and N alleles are based on three nucleotide substitutions in exon 2 and one base change (G or T) in an intron of glycophorin A locus. The latter single base change was also found among M alleles analyzed in this study, so that M allele appeared to be subdivided into M c and M T. All three alleles, M c, M r and N were identified clearly by RFLP or SSCP analysis following a single amplification. S and s alleles are based on one nucleotide substitution in exon 3 of glycophorin B gene. Genotyping of Ss blood group system was also explored by PCR-SSCP or ASPA analysis, and problems in the methods were discussed.

Systematic classification of alleles of the glycophorin A (MN blood group) gene

“…In our previous studies, M and N alleles were classified into six variations, provisionally called MN*M101, M102, M201, M202, N101 and N102, from the sequencing results of a total of 12,576 bp from the 5¢-flanking region to exon 7 of the GPA gene, with the exception of 30 kb of intron 1 (Akane et al 1997(Akane et al , 2000Mizukami et al 2002). These studies also revealed sequence data for new alleles M103, N103, N104 (N V ) and N201 (N 2 ).…”

“…MN serological phenotyping and genotyping were performed as reported previously (Nakayashiki and Sasaki 1996;Akane et al 1997;Li et al 1998;Sasaki et al 2000). Two new N alleles had been named temporarily N 2 and N v (Sasaki et al 2000) and are named N201 and N104, respectively, in this paper.…”

“…We have previously classified standard M and N alleles into several allele types based on differences in nucleotide sequence of the GPA gene (Akane et al 1997(Akane et al , 2000Mizukami et al 2002). The M allele was first divided into M G and M T alleles (Akane et al 1997). Further study allowed us to classify the M G , M T , and N alleles as M10X, M20X, and N10X, where the first letter (M/N), the second number (1/2) and the third to fourth digits (0X) represent antigenicity, major, and minor variations, respectively (Akane et al 2000;Mizukami et al 2002): M101 is the standard allele expressing M antigen, and alleles M102 and M201 are minor and major variants of M101.…”

“…We have previously classified standard M and N alleles into several allele types based on differences in nucleotide sequence of the GPA gene (Akane et al 1997(Akane et al , 2000Mizukami et al 2002). The M allele was first divided into M G and M T alleles (Akane et al 1997).…”

Allele-Specific, Inverse-PCR Amplification for Genotyping MN Blood Group

“…Glycophorin A (GPA), a sialoglycoprotein on the erythrocyte membrane, carries M and N antigens, based on two changes of the amino acid sequence at positions 1 and 5 (M, serine and glycine; N, leucine and glutamic acid). M and N alleles of the GPAgene are attributed to three nucleotide substitutions in exon 2 ( Figure 1) (10,18) and are determined by restriction fragment-length polymorphism (RFLP) analysis (1,6,12), single-strand conformation polymorphism (SSCP) analysis of the polymerase chain reaction (PCR) products (1) or allele-specific PCR amplification (ASPA) (4,8,13). MN genotyping by DNA analysis is informative for forensic identification and in several clinical situations where serological phenotyping is difficult or impossible (8), such as (i) in a patient transfused with large amounts of blood from various donors, (ii) a fetus at risk for hemolytic disease and (iii) a patient with autoimmune hemolytic anemia.…”