The antigens of the Knops blood group system (KN; ISBT 022) are located on the complement receptor 1 (CR1). All KN antigens identified so far are located in the Long Homologous Repeats LHR-C and LHR-D. Massively parallel sequencing (MPS) of all 39 exons of the CR1 gene in patients with suspected alloantibodies to KN antigens revealed several CR1 variants of unknown significance (VUS). The variants were located in different CR1 exons and phasing of the haplotypes remained unknown. We established long read Oxford Nanopore Technologies (ONT) sequencing for the LHR-C and -D region of CR1 for haplotype phasing.

The CR1 LHR-C and -D region was amplified by long range PCR with 5 overlapping PCR products from genomic DNA samples of 10 patients with at least 2 distant VUS. ONT sequencing was performed on MinION with R10.4.1 flow cells (Oxford Nanopore Technologies, Oxford, UK). After base calling and filtering the sequence data were aligned to the chromosme 1 sequence of the human reference genome GRCh38.p14 by using the Geneious Prime software (Version 2023.2.1; Biomatters Inc., Boston, MA, USA).

PCR amplification of the LHR-C and -D region was achieved from all samples with amplicon sizes of 10,601 to 12,797 bps. Long range ONT sequencing of the large amplicons confirmed the variants in the exons previously identified by MPS. Further variants were identified in the overlapping amplicon regions and were used for haplotype phasing of distant exon variants. In 6 samples we were able to identify the CR1 haplotypes including exon and intron variants with a distance of up to 41,699 bps. E.g., haplotype 1: c.3623A (exon 22, p.1208His, KN11) – c.4843A (exon 29, p.1615Ile, KN9) – c.5480C (exon 33, VUS); haplotype 2: c.3623G (exon 22, p.1208Arg, KN12) – c.4843G (exon 29, p.1615Val, KN10) – c.5480G (exon 33, VUS).

Long read ONT sequencing of large PCR products is suitable for haplotype phasing of the CR1 gene. The knowledge of the haplotypes could be useful especially in cases with alloantibodies against an unknown KN antigen and two ore more VUS in CR1. We were able to allocate 4 VUS to haplotypes of the LHR-C and -D region.

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