BLOOD-GROUP ANTIGENS RB-A, TR-A, AND WD(A) ARE LOCATED IN THE 3RD ECTOPLASMIC LOOP OF ERYTHROID BAND-3

BACKGROUND: Rb-a, Tr-a, and Wd(a) are three low-incidence blood group antigens that have not been assigned to a particular structure of the red cell membrane. Recent genetic and serologic data suggested erythro id band 3 as a possible carrier of these three antigens. STUDY DESIGN AND METHODS: Ten band 3 gene exons that encode the membrane domain of band 3 were screened for single strand conformation polymorphism (SSCP ). Exons displaying SSCP were cloned and sequenced; and the presence o f the mutations was verified by restriction digestion. RESULTS: Substi tutions 548 Pro-->Leu, 551 Lys-->Asn, and 557 Val-->Met, all located i n the third ectoplasmic loop of band 3, were detected in the subjects with Rb(a+), Tr(ac), and Wd(a+) red cells, respectively. The presence of the Rb-a and Wd(a) mutations was confirmed in additional carriers o f these blood group antigens. Chymotryptic cleavage at Tyr 553 and Tyr 555 abolished the agglutinability of Tr(a+) and Wd(a+) cells with the corresponding antisera, further demonstrating that the epitopes are l ocated in the third ectoplasmic loop of band 3. Similar quantities of mRNA corresponding to the two band 3 alleles, a normal pattern of red cell membrane proteins, and normal DIDS (4,4'-diisothiocyanato-stilben e-2,2'-disulphonic acid, disodium salt)-inhibitable sulfate flux were detected, which suggests that the mutations do not affect band 3 mRNA stability or band 3 protein expression and transport function. CONCLUS ION: Wd(a) and Rb-a, and tentatively Tr-a, can be assigned to the Dieg o blood group system.