Schnelle Methode zur Haplotypisierung von RHD/RHCE in voller Länge durch unspezifische long-range PCR Sequenzierung
Morgan Gueuning (Schlieren / CH), Gian Andri Thun (Schlieren / CH), Sonja Sigurdardottir (Schlieren / CH), Nadine Trost (Schlieren / CH), Kevin Engehausen (Schlieren / CH), Charlotte Engström (Schlieren / CH), Maja P. Mattle-Greminger (Schlieren / CH), Stefan Meyer (Schlieren / CH)
Resolving allele-specific sequences (haplotypes) for the RHD and RHCE gene can be highly challenging. Molecular techniques like cloning or cDNA sequencing have been utilized for this purpose, but they are not suitable in time-sensitive situations. Here, we introduce a rapid long-read sequencing method for haplotype reconstruction at the RH locus, exemplified by three distinct cases for which both serological and genotypic tests failed to offer unambiguous RHCE allele assignment.
Standard serological methods showed RH:1,2,3,4,5 phenotype in two donor cases. Additionally, PCR-SSP kits revealed heterozygous c.733C>G resulting in V+VS+ antigen expression. The third case was a patient urgently requiring transfusion; serotyped RH:1,2,-3,4,5 and molecular analyses showed presence of c.667G>T. To fully cover exons 1 to 10 from RHD and RHCE, we designed five generic primer pairs co-amplifying both homologous genes. PCR amplicon lengths ranged from 12.3 to 15.2 kb for RHD and 13.3 to 15.2 kb for RHCE. Overlaps of PCR amplicons comprised >1 kb and were chosen to lie within polymorphic regions to facilitate variant phasing. Pooled PCR products were barcoded allowing multi-sample Nanopore sequencing on MinION flow cells.
Long-range PCRs and library preparation were completed within half a day. Sequencing produced >20,000 reads in ~4 hours. Variant calls enabled full-length haplotype reconstruction in all three samples by identifying variants in overlapping regions. The c.733C>G variant was phased to different RHCE backgrounds: one donor exhibited a RHCE*ce allele (RHCE*01.20.01 | RHCE*04), while the other displayed the V+VS+ causing variant on a RHCE*Ce background (RHCE*02.30 | RHCE*03). Contrary to frequency-based expectations, the c.667G>T variant found in the patient was linked to RHCE*Ce (RHCE*02.22) associated with weak C rather than lying on RHCE*ceMO (RHCE*01.07, partial c), resulting in transfusion recommendation from a RH:1,-2,-3,4,5 donor.
Here, we present an amplicon-based Nanopore sequencing approach capable of rapidly and reliably obtaining complete RHD and RHCE haplotypes, at least in the absence of complex structural variation. This method is particularly well-suited for urgent scenarios where precise genetic allele data is critical for ensuring optimal blood provision. To the best of our knowledge, no other technique offers the ability to accurately infer RHD/CE haplotype sequences within a time frame of one working day.
No conflict of interest