RHD*Weak D Type 38: A Family Study

Abstract

AbstractAbstract 5155 Introduction:The RHD*weak D type 38 is produced by the single nucleotide change (833G>A) in RHD exon 6, carrying a Gly278Asp substitution in the transmembraneous RhD protein. This RHD allele shows a reduced expression of the D antigen on the red blood cell (RBC) surface and may be erroneously typed as D− by standard serologic methods, including the indirect antiglobulin test (IAT). The frequency of the RHD*weak D type 38 in Caucasians is reported to be 1. 5% in Ccee phenotype, while we have recently detected a relatively higher frequency of 2. 6% among Ccee phenotype in Brazil. In this study we performed a serologic and molecular analysis of three generations from a family with the rare RHD*weak D type 38. Study design and methods:The RBCs from 45 years-old woman blood donor tested D negative by routine serologic tests (Ccee phenotype) but were positive by the adsorption-elution technique. Further serologic investigation to establish the patterns of reactivity of the RBCs was preformed using anti-D Moabs IgM (clones HM10, P3X63, P3X21211F1, P3X21223B10; Diagast, Loos, France) and anti-D Moabs IgG/IAT (clones HM16, P3X35, P3X241, P3X249, P3X290; Diagast, Loos, France, and MS-26, ESD1; DiaMed, Latino América S. A.). Flow cytometric methods were employed to evaluate the D antigen densities, and molecular sequencing studies were done using a sequencing Kit (Big Dye Terminator v1. 1, applied Biosystems, Weiterstadt, Germany) and a genetic analyser (ABI 3100, Applied Biosystems, Foster City, CA, USA). Results:We found that the RBCs reacted negatively with 4/4 IgM anti-D, but showed weakly positive results with 4/7 IgG anti-D reagents. In face of such results we studied 12 members of her family and found that 6 more individuals tested D negative by standard serologic tests (Ccee phenotype) but were positive by adsorption-elution technique. The flow cytometry investigation of samples from the 7 subjects showed D antigen density in a range of 60 – 80 sites per cell and a Rhesus Index of 0. 1 – 0. 6. DNA sequencing analysis from 7 family members was performed showing that all descendants (siblings) had the same RHD*weak D type 38 genotype as the mother, also inherited by the grandson. Conclusions:The molecular analysis identified the presence of RHD*weak D type 38 in the three generations of a Brazilian family. The study revealed maternal inheritance (first generation) of the weak D type 38, with Ccee phenotype by her six children and the second generation showed paternal inheritance in a son. We also emphasize the significant prevalence of RHD*weak D type 38 in the Brazilian population, often showing a low antigen D density. Disclosures:No relevant conflicts of interest to declare.