Molecular basis for Rh(null) syndrome: identification of three new missense mutations in the Rh50 glycoprotein gene

Rh(null) is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rh(null) cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rh(null)(HT), RHAG harbored in exon 6 two G-->A transitions, GTT-->ATT and GGA-->AGA, which cause Val(270)-->Ile and Gly(280)-->Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rh(null)(WO), RHAG contained in exon 9 a single G-->T transversion, GGT-->GTT, which caused a Gly(380)-->Val missense change in TM12 segment. The G-->T transversion, which is located at the +1 position of exon 9, had also affected pre-mRNA splicing and caused partial exon skipping. Although both Rh(null) cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein.