Characterization of a new 0-allele of the ABO-gene

Propose: The genomic organization of the ABO gene which encodes a specific galactosyl-transferase includes 7 exons. The major variants of the Al antigen are A2. B, Ol and O2 and are defined by single nucleotide changes in the ABO gene. The O-allels disrupt the function of the galactosyl-transferase leading to the absence of the antigen, whereas the other nucleotide changes alter the galactosyl-residues added to the H-antigen. Until now, 15 allels expressing an A-antigen, 11 allels expressing a B-antigen and 8 allels resulting in a non-functional galactosyl-transferase (bloodgroup 0) were described. All sequence variations were found within exon 6 and 7. Here, we report the molecular characterization of a new O-allele identified incidentally through routine PCR-SSP genotyping of the ABO gene. Methods: DNA was isolated from EDTA blood samples of a healthy child and its mother in a case of paternity testing. Standard PCR-SSP proceedures were performed to analyze the polymorphisms 261del, C467T, G703A and G802A. For further characterization of the gene sequence, a 2 kb PCR amplification product was generated including exon 6, intron 6 and exon 7. After cloning into pGEM-Teasy vector, at least 50 single clones were sequenced and analyzed on an automated DNA sequencer. Sequence data were compared to the wild type ABO gene sequence as well as to the known variant sequences. Results: PCR-SSP genotyping of the four major polymorphisms of the ABO gene revealed a Ol/B genotyp in the DNA of the mother. The genotype of the child indicated allels with features of Ol, O2 and B allels. The entire genomic region including exon 6, intron 6 and exon 7 was cloned and subsequently sequenced using single clones. Approximately half of the clones revealed clearly the charactersitics of a O2-allele with A at position 802. All other clones analyzed showed the G-deletion at position 261 (exon 6) characteristic for Ol-allele and all characteristics of Ballels in exon 7, like G at pos. 526, T at pos. 657, A at pos. 703, A at pos., A at pos. 796, C at pos. 803 and A at pos. 930. The new allele obviously consists of exon 6 of a Ol allele and exon 7 of a B allele. Presumably, it resulted from meiotic recombination in intron 6 of the maternal Ol- and Ballele. Conclusions: We identified a new allele of the ABO gene. Because the allele contains the frame shift deletion at position 261, a truncated nonfunctional protein is encoded. The O-phenotye of the individual confirmed the O2/Onew genotype.