Integrated analyses reveal unexpected complex inversion and recombination in RH genes.

Phenotype D-- is associated with severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. It is typically caused by defective RHCE genes. In this study, we identified a D-- phenotype proband and verified RH phenotypes of other six family members. However, inconsistent results between the phenotypic analysis and Sanger sequencing revealed intact RHCE exons with no mutations in the D-- proband, but the protein was not expressed. Subsequent Oxford Nanopore Technologies whole-genome sequencing of the proband revealed an inversion with ambiguous breakpoints in the intron 2 and intron 7 and copy number variation loss in the RHCE gene region. Given that the RHCE gene is highly homologous to the RHD gene, we conducted a comprehensive analysis using Pacific Biosciences long-read target sequencing, Bionano optical genome mapping, and targeted next-generation sequencing. Our findings revealed that the proband had two novel recombinant RHCE haplotypes, RHCE*Ce(1-2)-D(3-10) and RHCE*Ce(1-2)-D(3-10)-Ce(10-8)-Ce(3-10), with clear-cut breakpoints identified. Furthermore, the RH haplotypes of the family members were identified and verified. In summary, we made a novel discovery of hereditary large inversion and recombination events occurring between the RHD and RHCE genes, leading to lack of RhCE expression. This highlights the advantages of using integrated genetic analyses and also provides new insights into RH genotyping.