Two new RHD alleles with deletions spanning multiple exons

• DOI: 10.1111/trf.16199
• Publication Date: 2020-11-26T03:00:20Z
• Authors: Antonella Matteocci; Jorge Monge‐Ruiz; Marianne Stef; Izaskun Apraiz; Lara Herrera‐del‐Val; Tommaso Mancuso; Katie Fennell; Monica Lopez; Yolanda Larizgoit...; Guido Nespoli; Montserrat Rubia‐...; Angela Collaretti; Luca Pierelli; Gorka Ochoa‐Garay
• Keywords: 03 medical and health sciences; Rh-Hr Blood-Group System; 0302 clinical medicine; High-Throughput Nucleotide Sequencing; Humans; genomic DNA; rhesus D antigen; Rho(D) antigen, 5' Rhesus box; allele; allele specific real time polymerase chain reaction; Exons; Polymerase Chain Reaction; Alleles; Sequence Deletion

AbstractBackgroundThe most common large‐deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large‐deletion RHD alleles reported to‐date consist of single‐exon deletions, such as RHD*01N.67 which includes exon 1.Materials and MethodsSamples from two donors with RhD‐negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele‐specific PCR, long‐range PCR, Sanger sequencing, and next‐generation sequencing assays.ResultsAllele‐specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next‐generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long‐range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5′ Rhesus box (upstream of the identity region for the 1‐to‐3 deletion, downstream of it for the 1‐to‐5 deletion), and end within introns.ConclusionsResolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5′ Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.