Charakterisierung auffälliger ABO Null-Allele mit Long-read Sequenzierung
Gian Andri Thun (Schlieren / CH), Morgan Gueuning (Schlieren / CH), Sonja Sigurdardottir (Schlieren / CH), Nadine Trost (Schlieren / CH), Samuel Koller (Schlieren / CH), Charlotte Engström (Schlieren / CH), Christoph Gassner (Triesen / LI), Maja P. Mattle-Greminger (Schlieren / CH), Stefan Meyer (Schlieren / CH)
We genotyped more than 36,000 blood donors for three ABO variants characterising ABO*O.01, O.02, and B-alleles using MALDI-TOF mass spectrometry with high-throughput capability. Discrepant results with serological values were further analysed by Sanger sequencing, but an exact allele determination was not always possible due to inability of phasing variants. To address this, we used long-read sequencing to elucidate the allelic background of unresolved samples by producing full haplotypes.
We analysed four cases with third-generation sequencing by Oxford Nanopore Technologies (ONT) that previously showed genotype-phenotype discrepancies. Those either pointed to silent ABO*A-alleles (n=2) or to suspected hybrid ABO*O.01-alleles harbouring variants defining ABO*O.02-alleles (n=1) or ABO*B-alleles (n=1). The entire ABO gene was amplified in two long-range PCRs of ~13 kb each with an overlap of almost 5 kb. Samples were barcoded and sequenced on a MinION flow cell according to standard protocols by ONT. Full-length haplotypes were aligned to ABO reference sequences and compared to the ISBT blood group allele table for ABO.
Our findings identified two new A1.01-derived null alleles: one with a combination of indels in exon 7 causing a frameshift (NM_020469.3:c.758_766del; NM_020469.3:c.768_769insG) and one containing the nonsense c.542G>A variant (defining O.06) along with four downstream variants representing O.09.01. Aligning the latter to ABO reference sequences suggested a rearrangement between A1.01 (up to end of exon 6) and O.01.02. The alignment for the third case pointed to a hybrid allele between O.01.02 and B (similar to O.01.24, but showing c.220C>T) with break point at beginning of intron 6. Finally, the last sample showed the O.01.57 allele whose variation downstream of exon 7 suggested a former recombination between O.01.01 and O.02.01 in exon 7.
Long-range PCR in combination with long-read Nanopore sequencing allowed straight-forward allele characterisation over the complete ABO gene, a cumbersome task with alternative technologies. We found three novel null alleles and argue that the increasing revelation of intronic information owing to long-read sequencing proves useful to categorise the high diversity found in ABO. Modern sequencing methods may soon trigger a more adequate representation of allelic variation in blood group genes.
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