RHCE genotyping using next generation sequencing: Allele specific reference sequences.

Background: The Rh blood group system (ISBT004) is encoded by two homologous genes, RHD and RHCE. Polymorphism in these two genes gives rise to 56 antigens, which are highly immunogenic and clinically significant. This study extended previous work on the establishment of RHD allele specific reference sequences using next generation sequencing (NGS) with the Ion Personal Genome Machine (Ion PGM) to sequence the complete RHCE gene.

Study design and methods: Genomic DNA (gDNA) samples (n = 87) from blood donors of different serologically predicted genotypes including R₁R₁ (DCe/DCe), R₂R₂ (DcE/DcE), R₁R₂ (DCe/DcE), R₂R_Z (DcE/DCE), R₁r (DCe/dce), R₂r (DcE/dce), R₀r (Dce/dce), rr (dce/dce), r'r (dCe/dce), and r″r (dcE/dce) were used in this study. The RHCE gene was amplified through overlapping long range-polymerase chain reaction (LR-PCR) amplicons and then sequenced with the Ion PGM. Data were analyzed against the human genome reference sequence build hg38 and variants were called.

Results: Referen variant allel VS. In addition to the RHCE reference alleles, different exonic single nucleotide variants (SNVs) were detected that encode known RHCE variant alleles including RHCE*Ce.09, RHCE*ceAR, and RHCE*ceVS.03. Numerous intronic SNVs were detected and compared from samples with different Rh genotypes, to determine their link to a specific Rh haplotype. Based on the exonic and intronic changes detected in different RHCE alleles, three RHCE reference sequences were established and submitted to Genbank (one for the RHCE*Ce allele, one for the RHCE*cE allele, and one for the RHCE*ce allele).

Conclusion: Intronic SNVs may represent a novel alternative diagnostic approach to investigate known and novel variants of the RH genes and the prediction of Rh haplotype.