RhD Immunization Despite Adequate Immunoprophylaxis: Role of Fc Gamma Receptor Gene Polymorphisms

Introduction: One of the most effective immunological interventions in clinical medicine is the prevention of hemolytic disease of the newborn by prophylactic Rh immune globulin (Rh-Ig) therapy. The administration of ante- and postnatal Rh-Ig has reduced the risk of RhD immunization in the Netherlands from 17% to a mere 0.31%, yet its mechanism of action is still unknown. To gain more insight into the possible working mechanism of the Rh-Ig prophylaxis we analyzed potential risk factors and genotyped all known IgG-Fc receptor (protein FcγR, gene FCGR) variants known to date, on a cohort of Dutch women who failed Rh-Ig prophylaxis and developed anti-D antibodies. Adequate Rh-Ig immunoprophylaxis was defined as an antenatal and postnatal prophylaxis of 1,000 IU (200 µg) in both current and previous pregnancies, according to the Dutch guidelines.

Material and Methods: Between 1999 and 2013 we identified 274 women who produced anti-D antibodies. Through a structured questionnaire we collected information about Rh-Ig prophylaxis and additional clinical data for potential risk factors. In 122 cases, adequate Rh-Ig prophylaxis was given, and clinical risk factors for fetal maternal hemorrhage (FMH) could be collected. Their clinical circumstances were compared to a control group of 339 randomly selected pregnant women. The Rh-Ig therapy failure of 57 of those women could not be explained through our risk factor analysis. From these 57 cases, DNA was obtained, and used for the FcγR-specific multiplex ligation-dependent probe amplification (MLPA) assay, identifying both single nucleotide polymorphisms and copy number variations in the FCGR locus. The results were compared to a control group of 200 healthy donors.

Results: A history of red blood cell transfusion (p=0.05) and caesarean section (p<0.0001) were identified to be independent risk factors for RhD immunization. All other described risk factors for FMH such as miscarriage, termination of pregnancy, or invasive diagnostic procedures, requiring an additional Rh-Ig dose according to the guidelines, were not found to increase the risk of immunoprophylaxis failure. RhD-immunization due to caesarian section or red blood cell transfusion accounted for 53% of our cohort, suggesting an alternative explanation for the production of Rh-Ig alloantibodies in the remaining 47% of the cases - despite adequate amount of prophylaxis given in current and previous pregnancies. We therefore postulate the existence of a genetic variation that puts women at increased risk for RhD immunization during pregnancy. To test this hypothesis we analyzed the genetic variation in the FCGR locus and found a significantly (p=0.02) increased prevalence of the FCGR2 -ORF, expressing a functional copy of the activating FcγRIIc, which is otherwise a pseudogene. Strikingly, the prevalence of the 2B.4-promotor haplotype of the FCGR2B gene, associated with a 1.5 fold increase of the inhibitory FcγRIIb, was strongly (p=0.0001) increased.

Conclusion: Caesarian section and red blood cell transfusion are risk factors that increase RhD immunization during pregnancies, accounting for about half failed Rh-Ig prophylactic cases. Genetic variation in the FCGR-gene might be a possible explanation for increased immunization risk. In our cohort we encountered a significantly increased frequency of individuals expressing FcγRIIc, along with a polymorphism encoding for a higher expression of the inhibitory receptor FcγRIIb, suggesting these genes to influence immune responses to RBC in a manner previously unrecognized.