Integrated analyses reveal unexpected complex inversion and recombination in RH genes

Abstract

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 6 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 whole-genome sequencing by Oxford Nanopore Technologies of the proband revealed an inversion with ambiguous breakpoints in 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 2 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, to our knowledge, a novel discovery of hereditary large inversion and recombination events occurring between the RHD and RHCE genes, leading to a lack of RhCE expression. This highlights the advantages of using integrated genetic analyses and also provides new insights into RH genotyping.

Graphical abstract

Figure 1.

Pedigree tree with RH haplotype heredity.

Figure 2.

The expression of RBC membrane RhD and RH-related antigens from D-- family members. The expression levels followed a decreasing order of RR>Rr>D^v>D-. (A) RhD antigen of D-- family members. (B) RH-related antigen of D-- family members. (C) Histogram of the results in panel A. (D) Histogram of the results in panel B.

Figure 3.

Genome structural variation analysis of the proband. (A) ONT read coverage in the RH gene region of the D-- proband. (B) NGS read coverage in the RH exon region of the D-- proband.

Figure 4.

Reference RH and SV haplotype models. The purple border arrows indicate the joining point of homologous recombination; red border arrows indicate the joining point of nonhomologous junction; yellow filled arrows indicate the same joining points in different SV. (A) Reference RH haplotype model. (B-C) Models of the SV haplotype of the father. (D-E) Models of the SV haplotype of the mother.

Figure 5.

SV haplotype models of the proband generated by using Bionano OGM. (A) Models of the SV haplotypes of the proband (the red border boxes represent the SV regions). (B) Models of the distribution of Bionano labels between the reference genome and the sequenced sample in the RH gene region (the top figure presents the reference genome and the vertical lines on the axis indicate Bionano labels. The middle visualization depicts the Bionano analysis between the reference genome and the sequenced sample, with lines connecting the same label. The bottom part shows blue lines linking the reference genome with the corresponding exons of the proband. The light red triangle in the last 2 figures indicates the inserting region).

Figure 6.

RH locus landscape and breakpoint sequencing. The representative introns and exons are shown as lines and boxes below the coordinate axis, respectively; the arrow on the last exon represents the genic orientation. RH boxes are shown as purple boxes below the axis. Twenty-nine heterozygous SNV markers were developed from HiFi cluster reads and involved in assembling and phasing haplotypes of this locus (strings in each gray box give the genomic coordinate, reference to alternative alleles of corresponding SNV marker). (A) Nineteen amplicons were designed to target the whole region of this locus and identify breakpoints therein (amplicons are shown as brownish red bars above the axis, and the recognized nonhomologous end-joining breakpoints and homologous recombination breakpoint regions are marked in lime and magenta/red, respectively). (B) The breakpoint of CE8-CE3 fusion tested via Sanger sequencing (inversion intervals are shown as arrows above the base pairs).