Background/Case Studies: The RHD genetic diversity is high, with more than 600 alleles reported to date. Most alleles have underlying variants that encode amino acid changes, with a few affecting splice sites and transcription factor binding sites. We investigated four samples referred with weak D typing.
Study
Design/Methods: Blood samples (S1-S4) were collected from four patients. Serologic testing was done by standard tube method with commercial Anti-D reagents: Ortho BioClone, Immucor Gamma-clone, Bio-Rad Seraclone Blend. Genomic DNA was isolated from WBCs (QIAGEN). Targeted genotyping was done with RHD BeadChip (Immucor) and in-house assays. RHD exons and flanking intron regions were amplified and Sanger sequenced. ASP-PCR, followed by sequencing, was used to phase the new variant in sample S1.
Results/Findings: Table 1 summarizes the samples (S1-S4), race/ethnicity, serology, and molecular findings. RBCs of S1, a 34-year-old female, reacted weakly at IS and moderately at IAT with Ortho and Bio-Rad and reacted strongly at IS and IAT with Immucor Anti-D. Targeted genotyping found RHD in trans to RHD*03N.01 [hybrid RHD*DIIIa-(4-7)-D]. Sequencing identified heterozygosity for variant c.212G>A (p.Arg71Lys), confirmed to be in trans to RHD*03N.01 by sequencing of ASP-PCR products. RBCs of S2, a 27-year-old Hispanic female, reacted moderately at IS and strongly at IAT with Ortho, Bio-Rad and reacted strongly at IS and IAT with Immucor Gamma-clone Anti-D. By targeted genotyping, the sample was RHD hemizygous. Sequencing found c.1091T>C (p.Leu364Pro). RBCs of S3, an infant boy, were non-reactive with Ortho and Bio-Rad and reacted microscopically at IS with Immucor Gamma-clone and reacted strongly at IAT with all Anti-D. Targeted genotyping found c.787G>A (p.Gly263Arg) on a RHD*DAU3 background, and in trans to RHD*03N.01 [hybrid RHD*DIIIa-(4-7)-D]. Sequencing confirmed these results. RBCs of S4, a pregnant (G8P1) White female, reacted weakly with Ortho and Bio-Rad and strongly with Immucor Gamma-clone at IS and reacted strongly at IAT with all Anti-D. Sample was RHD hemizygous by targeted genotyping. Sequencing identified a c.1154-4A>T near the intron 8 acceptor splice site. Conclusions: We report four novel variant alleles in samples with serologic weak D phenotype: RHD*212A, *1091C, *DAU3(787A), and *1154-4T. Interestingly, c.787G>A by itself has been previously reported in Italian, Chinese and Japanese individuals, whereas the variants in S3 are typically found in those identifying as Black. Although intron position -4 has rarely been associated with an altered phenotype, an effect for c.1154-4A>T is supported by the fact that it is the only variant found in the sequenced regions and by variant c.940-4A>C in intron 6, reported to lead to a weak D phenotype.
Importance of research: Weak or discrepant D typing can pose a challenge for decisions of RhIG administration and transfusion of patients. With limitations of serology testing, it is important to investigate these samples by RHD genotyping. We present four new RHD alleles associated with serologic weak D phenotype.
