Novel missense variants in the RHAG gene that result in Rh^mod phenotype

Identification of variants causing Rh deficiency phenotypes is important to allow safe transfusions and prevent hemolytic disease of the fetus and newborn. During preoperative screening for pseudomyxoma peritonei, a patient presented with suspicion of Rh-deficiency syndrome. The effect of these variants on the expression of Rh and Rh associated proteins was studied.

Serotyping, MLPA genotyping, and next-generation sequencing were performed on the RBCs of the proband and their family members. The effect of the variants on membrane expression of RhAG, Rh proteins, as well as Rh complex membrane proteins was evaluated using flow cytometry and western blot. Transient transfection experiments were conducted to assay the consequences of the RhAG variants on RhAG, RhD, and RhCE membrane expression.

Two novel missense variants in RHAG, RHAG:c.172C>T and RHAG:c.242G>A were identified resulting in an Rh^mod phenotype. The novel RHAG variants decreased RhAG expression with a concomitant reduction in expression of Rh proteins and CD47 while a slight increase in band3 expression was observed in the proband. A reduction of CD44 expression was also observed. Transfection of c.172C>T did not lead to a lower RhAG expression, while in contrast, expression of c.242G>A was significantly decreased compared to RhAGwt. Both led to a reduction of RhD and RhCE expression. Co-transfection of RHAGwt with c.172C>T or c.242G>A led to a significant reduction in RhD and RhCE expression.

Two novel RHAG variants leading to Rh^mod phenotype were identified. A decrease in CD44 expression not previously associated with this phenotype was observed.