Outliers in RhD membrane integration are explained by variant RH haplotypes

Area of Science:

• Immunogenetics
• Molecular biology
• Transfusion medicine

Background:

• Variations in multipass transmembrane proteins can alter membrane integration.
• D antigen density serves as a model for studying these effects.
• D antigen expression is often determined by a single allele due to frequent hemizygosity.

Purpose of the Study:

• To systematically characterize variant D antigen density.
• To investigate the molecular basis of D antigen densities outside the typical distribution.
• To identify novel RHD alleles and understand their impact on D antigen expression.

Main Methods:

• Flow cytometry was used to determine D antigen density distribution in 530 CcDee, 475 ccDEe, and 514 ccDee samples.
• Molecular investigation of samples with atypical D antigen densities.
• Quantitative polymerase chain reaction (qPCR) to assess RHD and RHCE allele dosage.

Main Results:

• Three new RHD alleles (Weak D type 19, Weak D type 20, and partial D DYU) were identified in samples with decreased D antigen density.
• Increased D antigen density was observed in samples lacking a hybrid Rhesus box, suggesting RHD homozygosity.
• Some samples predicted as heterozygous showed a large RHD to RHCE allele ratio, with one case revealing an RHD-CE hybrid transcript.

Conclusions:

• Distinct protein variants underlie unusual RhD protein integration and D antigen density.
• Weak D antigen expression is consistently linked to variant RHD alleles.
• Elevated D antigen density is often due to homozygous alleles, potentially confounding zygosity tests based solely on gene copy number.