(P-IG-11) Apparent RHCE phenotype discrepancy in a mother and child due to a shared RHCE*CE-D(3-9)-CE allele

Background/Case Studies: Samples from a mother of Portuguese descent and her newborn daughter were referred for RH genotyping following the discovery of apparently incompatible RH phenotypes. During her pregnancy, the mother had a negative antibody screen and her red blood cells (RBCs) typed D+C-E-c+e+ by automation (Qwalys, Diagast). She delivered at 37 weeks of pregnancy. Because the newborn had dextro-transposition of the great arteries and required surgery, her RBCs were typed, and were found to be D+C-E+c+e- by automation (Innova, Ortho). There was no history of gamete donation. Whole blood samples were referred to investigate the E/e discrepancy between mother and child by RH genotyping.

Study

Design/Methods: DNA was extracted from white blood cells. RHCE BeadChip (Immucor) to detect common variants and quantitative multiplex PCR of short fluorescent fragments (QMPSF) to detect hybrid genes were performed for both samples.

Results/Findings: The mother’s sample was homozygous for c.676G/G and c.733G/G by the RHCE BeadChip, predicting E-e+ phenotype and consistent with the observed phenotype. The newborn’s sample was homozygous for c.676C/C, predicting a E+e- phenotype and confirming the discrepancy. An additional, heterozygous c.48C/G was found in both samples, suggesting a hybrid allele expressing neither E nor e antigens and encompassing at least exon 5. QMPSF confirmed this hypothesis by showing that mother and daughter shared the hybrid allele RHCE*CeN.08 [RHCE*Ce-D(3-9)-CE]. QMPSF also found that mother and daughter each had two RHD genes.

Conclusions: The mother’s RHCE alleles were RHCE*01.20.01/RHCE*CeN.08 and the daughter’s were RHCE*03/RHCE*CeN.08. RHCE*CeN.08 is considered a null allele with regards to RHCE antigen expression, resolving the apparent phenotype discrepancy between mother and child. However, the RHCE*CeN.08 allele encodes the same protein as RHD*01.01 (DUC), that is RhD with p.Trp16Cys, and would be expected to type D+ in a sample with no RHD gene. The newborn required transfusion of 2 RBC, 1 platelet and 3 plasma units for her heart defect surgery, with no adverse events reported.

Importance of research: We report the investigation of an immunohematology case confounded by routine genomics testing. Its resolution required quantitative multiplex PCR of short fluorescent fragments (QMPSF) to detect hybrid genes, a simple technique that is unfortunately not available in many genomics labs. Hybrid genes may be suspected but are often difficult to demonstrate.