An RHD gene variant, common in southern Chinese Han, involves a short 688 bp deletion and a long 21.8 kb insertion in...

Accurate typing of the D antigen is critical to provide guidance for blood transfusion and safe management of Rh-incompatible pregnancies. RHD genotyping assays have been widely adopted to improve D variant detection, especially for common RHD alleles with missense variants and RHD-CE-D hybrids. Few structural variants of the RHD gene are known to involve large fragments of non-RH sequences, despite causing phenotype-genotype discrepancies.

We had previously detected reduced copy numbers of RHD exon 10 in Chinese Han. Their RHD genes were further analyzed using long-read sequencing of RHD and whole-genome long-read sequencing using PacBio platform. The population frequency and practical relevance were evaluated, such as effects on D antigen expression and accuracy of routine RHD genotyping assays.

A novel structural variant of the RHD gene, dubbed RHD-CE-TMEM50A-D, had distinct features: a short deletion of the non-coding region of RHD exon 10 (688 bp del), a large insertion (21.8 kb ins) involving the inversion of non-coding region of RHCE exon 10 (174 bp) and exon 7 to intron 2 of TMEM50A gene (21,648 bp), and several single nucleotide substitutions. The large delins fragment was firstly found in 9 individuals, further representing 4.1% of random D+ donors (40/982), 2.9% of D-C+/E+ individuals who carry the non-functional RHD*01N.04 (6/205), and 3.4% of weak/partial D individuals (7/207) who carry RHD*DFR2 and RHD*DVI.3, but 0% in a different cohort of Asian-type DEL individuals (0/205). The large delins fragment located in non-coding region of RHD exon 10 of four different alleles did not significantly change the D antigen expression but caused inconclusive results in several routine RHD genotyping assays.

The novel RHD-CE-TMEM50A-D allele represented approximately 1 in 49 chromosomes among the southern Chinese Han population. The variations of these RHD alleles involve the replacement of some RHD-specific sequences by the corresponding RHCE-specific sequences in the non-coding region of RHD exon 10, commonly used for RHD-specific primers design in genotyping assay. Hence, red cell genotyping assays, if applied to individuals of East Asian heritage, should recognize this relatively common allele to avoid RHD allele dropout.