The novel c.1227+2T&gt;C variant in <scp><i>RHD</i></scp> results in the serological D‐negative phenotype

The Rh blood group system, one of the most complex of the 47 known blood group systems,1 includes the highly immunogenic D antigen, which is vital in transfusions and pregnancy owing to the risk of anti-D antibody formation. Over 500 RHD allele variants, including single-nucleotide variants and gene conversions, have been identified globally. Researchers continue to explore these variants using integrated molecular and serological methods. In this study, we report a novel c.1227+2T>C variant in the RHD gene, which resulted in a serological D-negative phenotype in the patient. The D antigen status was evaluated using two different monoclonal IgM anti-D antibodies (clones MS-26 and D175-2) provided by Sanquin (Amsterdam, the Netherlands) and Dominion Biologicals Ltd (Nova Scotia, Canada) through tube testing methods. For negative results, an indirect antiglobulin test was conducted using a polyspecific anti-IgG anti-D antibody (Shinyang Diagnostics, Siheung, Korea) with a microcolumn card (Ortho BioVue® System, Ortho-Clinical Diagnostics, Pencoed, UK). RhCE phenotyping was performed using the tube method, with antibodies against each antigen (Shinyang Diagnostics). Genomic DNA was extracted from peripheral blood using a MagNA Pure 96 DNA Isolation Kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Multiplex PCR targeting the promoter, intron 4, exon 7, and exon 10 of RHD was performed using a GeneAmp™ PCR System 9700 (Applied Biosystems, Foster City, CA, USA). Sanger sequencing of the RHD gene was conducted using an ABI Prism 3730xl genetic analyzer (Applied Biosystems, Foster City, CA, USA - Applied Biosystems) with a BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The primers for PCR and Sanger sequencing were designed in-house for routine clinical testing.2 NM_016124.6 (GRCh37) was used as the reference sequence. The in-silico prediction was performed using SpliceAI, with a cutoff of Δ Score >0.2 to define potential splice-altering variants. The sample for analysis was obtained from an 84-year-old female with uremic encephalopathy who underwent a transfusion test for disseminated intravascular coagulation. Serologic tests yielded negative results for both RhD phenotyping and the weak D test. The RhCE phenotype was “Ccee.” Anti-D adsorption/elution tests were not performed. To resolve the molecular basis of the serological D-negative phenotype in this case, we performed RHD genotyping in accordance with our institutional policy. RHD genotyping is recommended when both the weak D test is negative and the RhCE phenotype differs from “ccee.” Multiplex PCR revealed the presence of all targeted exons and introns. Sanger sequencing revealed a homozygous c.1227+2T>C variant, which is a novel variant located in intron 9 of RHD. No other variants were detected in the exons or exon-intron boundaries. The c.1227+2T>C variant has not been reported in the public population database (gnomAD v4.1.0) or the Korean allele frequency database (KRGDB 1722).3 In-silico prediction showed a high probability of splicing change, with SpliceAI Δ scores of 0.86 for acceptor loss type and 0.90 for donor loss type. The delta scores obtained using SpliceAI suggest that this novel variant is likely to have a major functional effect on splicing. This finding is similar to that of the RHD*DEL1 (also known as Asian-type DEL) allele, which exhibited a maximum SpliceAI Δ score of 0.84. As a splice site variant, the c.1227+2T>C variant is predicted to result in a shortened mRNA transcript due to exon 9 skipping caused by the loss of the donor splicing site, similar to the c.1227G>A variant.4 This result ultimately represents an effect similar to RHD*DEL1, known as Asian-type DEL.5 However, there is currently insufficient evidence to confirm that the c.1227+2T>C variant allows for safe transfusion of D-positive blood. Therefore, transfusion of D-negative blood products is recommended in non-emergency situations or when there is no shortage of D-negative blood products. Unlike the c.1227G>A variant of Asian-type DEL, which is located at the last base of exon 9, the variant identified in this patient affects the invariant +2 position of the canonical donor splice site. This position is highly conserved across human exons, as shown in a comprehensive genome-wide analysis of splice site conservation6 Numerous splicing variants have been classified as DEL types and registered with the International Society of Blood Transfusion (ISBT) RHD blood group allele table.1 Notably, five variants at the canonical exon–intron boundary, the +2 site of the RHD gene (c.486+2T>A, c.801+2T>A, c.939+2T>A, c.1073+2T>A, and c.1073+2T>C), have been reported to date. These variants exhibit distinct phenotypes and are associated with either the DEL type or a D-negative phenotype.7 Additionally, there have been reports suggesting that the functional impact of these variants may vary depending on the type of mutation.8 Furthermore, the splicing effects of several alleles have previously been validated by functional studies.9 Therefore, to determine the function of the novel c.1227+2T>C variant, further evaluation is required, including RNA sequencing and additional tests to confirm the absence of anti-D antibodies following the transfusion of D-positive blood. In conclusion, this study presents a genetic analysis of a Korean patient with a novel c.1227+2T>C variant in the RHD gene. This variant is located at a canonical splicing site in intron 9, and it may affect mRNA processing through exon skipping, ultimately resulting in a serological D-negative phenotype. The authors have disclosed no conflicts of interest.