Characterization of two novel RHD mutations inducing aberrant D expression

The RH blood group system is encoding 56 different antigens (31-MAR-2022, ISBT) carried on two glycoproteins (RhD and RhCE). Gene mutations result in variant proteins which can alter the expression of RH antigens. While the number of known RHD alleles is growing, the serologic characterization of the expressed phenotype is getting increasingly challenging. Here we used Sanger sequencing to analyze the molecular background of two novel RHD mutations, which induced aberrant Rhesus D-expression.

Blood samples of two patients were sent to our immunohematology laboratory for analysis. RH phenotypes were determined by standard blood group serology (D-Screen Panel Diagast, Loos, France). Genotyping was carried out by PCR-SSP (RBC-Ready Gene CDE and RBC-Ready Gene D weak, Inno-Train, Kronberg; Partial D-Type, BAG Diagnostics, Lich). Sanger sequencing on an ABI Prism 310 (Applied Biosystems, Weiterstadt) was applied for further molecular analysis. Custom primers specific for RHD were used for amplification and sequencing reactions of all ten exons including short flanking intron regions.

Serological results implied a weak D or partial D phenotype. Further analysis using two different PCR-SSP Kits showed the reaction pattern of RHD*01. Common weak-D was excluded with further SSP-PCR. Sanger sequencing determined an RHD*c.523T>A, inducing a p.Phe175Ile exchange in the first patient. The other patient carried a RHD*c.266A>G (p.Gln89Arg). Since serological testing revealed an aberrant D-expression in both samples, it can be inferred that both mutations are located on the allele coding for RHD*01. According to Wagner et al. (Blood, 1999), the amino acid exchanges should be located within the third membrane and the sixth membrane passage of the RhD molecule, respectively. This could explain the atypical serological result.

Both mutations led to the detection of altered D-expressions. Sanger sequencing made it possible to clarify the presence of a D variant and to identify these novel mutations and therefore still poses a beneficial tool for the correct analysis of atypical serological results. Neither of the mutations is listed in the ISBT allele tables thus far. The nucleotide sequences of the new mutations have been submitted to the GenBank data base (GenBank accession numbers OQ451803, OQ469491).

No conflict of interest.