Characterization of two novel ABO splice site variations preventing blood group A and B expression

Background

The ABO locus is highly polymorphic with almost 200 alleles that encode altered phenotypes. Gene mutations result in functionally modified glycosyl transferases that can change the expression of ABO antigens. Rare mutations can push the limits of conventional serology or PCR-SSP methods. Analysis by Sanger sequencing can help to resolve complex cases. In this work, we analyzed the molecular background of two novel splice site variations, which prevent the expression of blood group A and B.

Methods

ABO phenotypes were determined by standard blood group serology (BioRad, DiaMed GmbH, Cressier, Schweiz). Genotyping was carried out by PCR-SSP (RBC Ready Gene ABO Subtype, Inno-Train, Kronberg). Sanger sequencing on an ABI Prism 310 (Applied Biosystems, Weiterstadt) was applied for further molecular analysis. Custom primers specific for ABO were used for amplification and sequencing reactions of all seven exons including short flanking intron regions.

Results

Blood samples of two patients showed inconsistent results between blood group and reverse typing. Further analysis with PCR-SSP could not explain the aberrant serological results. For safety reasons, both patients were assigned to blood group O. Sanger sequencing determined a heterozygous ABO*204 -2g>a located two nucleotides downstream of exon 1 in patient one and a heterozygous ABO*c.28+3ins t three nucleotides upstream of exon 5 in patient two. Both variations qualified as splice site mutations that affect the transcription of the antigen. Since serological testing revealed a weakened A, respectively B, expression in the two samples, it can be inferred that the mutations are located on the alleles coding for ABO*A1.01 and ABO*B.01.

Conclusion

Both mutations led to the detection of altered blood group A and B expressions, resulting in Ax/Ael and Bweak/Bx/Bel phenotypes. Sanger sequencing allowed the identification of these novel mutations and therefore still is beneficial in advanced analysis of atypical serological results. Neither of the mutations is listed in the ISBT allele tables thus far. The nucleotide sequences of the new mutations have been submitted to the GenBank data base (GenBank accession numbers OQ469492, OQ595206).

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