Two new RHD alleles with deletions spanning multiple exons

Area of Science:

• Genetics
• Molecular Biology
• Immunology

Background:

• The RHD gene determines the D antigen in RhD-positive individuals.
• Large deletions in the RHD gene are a known cause of RhD-negative status.
• Previously identified large-deletion RHD alleles include RHD*01N.01 (entire coding sequence deleted) and RHD*01N.67 (exon 1 deletion).

Purpose of the Study:

• To determine the RHD genotypes of two donors with RhD-negative serology who had inconclusive results from RHD genotyping arrays.
• To characterize novel large-deletion RHD alleles.

Main Methods:

• Genomic DNA analysis using allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing.
• Quantitative next-generation sequencing to confirm exon deletions.
• Identification of deletion breakpoints.

Main Results:

• One sample showed deletion of RHD exons 1-3, and the other showed deletion of exons 1-5.
• Next-generation sequencing confirmed these deletions and the absence of an RHD gene in trans.
• Long-range PCR and Sanger sequencing identified deletion breakpoints within the 5' Rhesus box and introns.

Conclusions:

• Unclear genotyping results can lead to the discovery of new RHD alleles.
• The 5' Rhesus box may be a recombination hotspot for large RHD deletions.
• Characterization of these novel alleles improves RHD genotyping accuracy.