Routine testing for RHD*01W.1, RHD*01W.2 and RHD*01W.3 subtype alleles is dispensable in Switzerland

Background

Alleles RHD*01W.1, RHD*01W.2 and RHD*01W.3 are defined by single nucleotide variants (SNVs) which lead to reduced RH1 antigen expression. Anti-RH1 alloimmunization has not been reported for individuals carrying these alleles. Subtypes with additional SNVs have been described and may cause a risk for alloimmunization. Therefore, we developed sequence specific primer (SSP)-PCRs to genotype individuals with known RHD*01W.1/.2 and .3 alleles to elucidate the frequency of these subtypes.

Methods

Blood donor and patient samples from 2019-2022 (four years) showing a weak RH1 phenotype were genotyped using the RBC-FluoGene D weak/variant kit (inno-train, Germany) identifying SNVs c.809T>G (RHD*01W.1), c.1154G>C (RHD*01W.2) and c.8C>G (RHD*01W.3). In-house developed multiplex SSP-PCR assays were used to further screen for subtypes defined by the presence of the SNVs c.52C>G (RHD*01W.1.1), c.712G>A (RHD*01W.1.2), c.667T>G (RHD*01W.1.3), c.455A>C (RHD*62), c.301T>A (RHD*01W.2.1), c.916G>A (RHD*01W.2.1) and c.178A>C (RHD*01W.3.1). The Human Growth Hormone gene (HGH) served as DNA extraction and amplification control. Amplicons were analyzed by capillary gel electrophoresis (Qiagen, Germany).

Results

A total of 714 samples carried c.809T>G of which two samples revealed the presence of the RHD*01W.1.1 defining substitution c.52C>G. Two samples belonged to the subtype RHD*62 due to c.455A>C. One sample showed the presence of c.667T>G indicative of RHD*01W.1.3. However, further analyses revealed that this SNV was detected in addition to c.602C>G rendering the presence of alleles RHD*01W.1 and RHD*09.03.01 more likely, rather than the much less probable combination RHD*01W.1.3 and RHD*01W.40. Furthermore, we identified 274 and 157 samples carrying the SNVs for RHD*01W.2 and RHD*01W.3, respectively. In none of these samples the screen revealed any additional SNVs.

Conclusion

Subtypes of alleles RHD*01W.1, RHD*01W.2 and RHD*01W.3 are extremely rare. Only four out of 1145 samples (0.35 %) carried additional SNVs, all of them associated with the SNV c.809T>G. Moreover, alloimmunization in individuals carrying the investigated subtype alleles has not been reported to the best of our knowledge. Considering the rareness of subtypes of alleles RHD*01W.1, RHD*01W.2 and RHD*01W.3, screening for these subtypes will not be proposed to be performed on a routine basis.

Offenlegung Interessenkonflikt:

The authors have no conflict of interest to declare.