(P-IG-8) An Observation of „de Novo“ Occurred New Mutation in the ABO*A1.01 Allele – Insertion of an Additional G in the Region c.811-817, Causing Loss of Serological Expression of a Antigen

Background/Case Studies: ABO is the clinically most important blood group system, but genotyping still remains a challenge. In addition to the major ABO alleles, numerous variants responsible for the weakening or loss of expression of A and B antigens have been identified. Here we describe a rare observation of “de novo” occurring new mutation on the ABO*A1.01 allele – insertion of an additional G in the region c.811-817, causing loss of serological expression of A antigen. A similar one on the same allele was described in an individual from Papua New Guinea, on the A1.02 allele in a Taiwanese family, and on the B1.01 allele in a blood donor from Germany. These mutations on A alleles have not been listed in the ISBT table so far.

Study

Design/Methods: Blood samples from a proband and her parents were investigated. Standard serological techniques were used (manual technique in gel cards and agglutination test on Galileo ECHO analyzer), in proband the forward and reverse typing was additionally performed with enzyme (bromelin) treatment. The ABO locus was analyzed by RBC-FluoGene ABO Basic, RBC-FluoGene^NX ABOplus, RBC-NGStype CORE on the Illumina platform and with the long-range sequencing approach of RBC-NGStype full ABO (in development) on Oxford Nanopore Technology (ONT). DNA analysis of 20 short tandem repeats (STRs) polymorphisms and sex-specific amelogenin was performed in the patient and her parents using the PowerPlex® 16 HS System and PowerPlex® CS7 System.

Results/Findings: The patient’s RBCs were initially typed as group O but she lacked anti-A in her plasma, both with untreated and bromelin treated RBCs. Father’s typing was normal A1 and mother’s normal O. The ABO genotype of the proband was ABO*A1/O.01, in RBC-FluoGene ABO Basic, consistent with normal group A1. The same sample in RBC-FluoGene^NX ABOplus showed positive reaction for SNP 815insG but the software did not assign any known matching pattern. Using RBC-NGStype CORE and ONT long-range sequencing a G insertion was detected in exon 7 of the ABO*A1.01 allele in c.811-817, exact position cannot be assigned since there is a poly-G-sequence. This mutation was not present in both parent´s DNA. The biological parentage was confirmed. The analysis of STR polymorphisms revealed a single cell line population in the peripheral blood of the patient.

Conclusions: Novel mutation on ABO*A1.01 allele (not listed in ISBT table) was observed as a „de novo“ occurred genetic event in a common hot spot poly-G-sequence in exon 7. This mutation results in a frame shift that leads to a 37-amino acid longer polypeptide which dramatically affects the A antigen expression (similarly to that of the Ael01 allele), which was undetectable by serological methods. The absence of anti-A isoagglutinin however suggests some A-transferase activity should be present in the proband. Further studies are needed to clarify this phenomenon.