Specific amino acid substitutions cause distinct expression of JAL (RH48) and JAHK (RH53) antigens in RhCE and not in RhD

Westhoff and colleagues1 and Hustinx and coworkers2 have recently reported a total of three molecular causes for the low-prevalence Rh antigen JAL (RH48). Quantitative weakening and qualitative effects are documented for these variant RhCE proteins. Similar serological effects have been observed in the JAHK+ (RH53),3 ceSL,4 and RhCe(S122P)5 variants of the RhCE protein. We have observed 4 RhD variants harboring similar amino acid substitutions at positions 114 or 122 (Table 1). Table 1 RHD alleles encoding amino acid substitutions at positions 114 and 122 Most Rh antigens are known to be associated with molecular variants of either the RHD or RHCE gene. However, distinct Rh antigens, like c (RH4), G (RH12), Rh32 (RH32), Evans (RH37) and FPTT (RH50), can be expressed by RhD and RhCE variants (Table S1, available online). We collated all low-prevalence Rh antigens caused by single nucleotide substitutions and identified pairs of RHD and RHCE alleles that harbor identical substitutions (Table S2, available online).1–4,6 Only amino acid substitutions at positions 114 and 122 were currently found to fulfill both criteria. In RhCE these substitutions cause JAL or JAHK antigen expression. Hence, we assessed the possible expression of JAL and JAHK by RhD proteins harboring similar amino acids at positions 114 or 122. A DVII sample harboring a substitution at 110 was used as negative control. All RhD samples with amino acid substitutions at position 114, weak D type 17, type 25 and type 47, were negative for the JAL antigen (Table 2). The CeMA and Rhce(R114Q) control samples were JAL positive, as expected. Because the antigen density of weak D type 17 is very low (Table 1), we corroborated its lack of JAL expression by a negative adsorption-elution test (not shown). Table 2 Serologic results of RhD and RhCE variants with amino acid substitutions at positions 114 and 122 The weak D type 54 sample with an amino acid substitution at position 122 was negative for the JAHK antigen (Table 2). The RhCe(S122L) control sample was positive with the 3 anti-JAHK sera, as expected. ceSL has been reported to be JAHK negative.4 We confirmed this result with the original red blood cell (RBC) samples and the anti-JAHK P4.53A serum used previously. However, the two anti-JAHK sera of greater titer revealed that ceSL samples were in fact JAHK positive (Table 2). Both RhCe(S122P) samples were JAHK negative with all 3 sera. The two sera P4.53B and P4.53C were identified from a series of 138 samples collected from pregnant women with a positive antibody screen.7 Both women had neither been transfused nor were their newborns or the putative fathers positive for the JAHK antigen.7 Hence, anti-JAHK is possibly a rather frequent, naturally occurring antibody associated with pregnancy. The titers of anti-JAL (Table 2) determined with CeMA and Rhce(R114Q) differed in congruence with the known weaker expression of JAL in the Rhce variants Rhce(R114Q) and ces(340) compared to the RhCe variant CeMA.2 In contrast to Rhce(R114Q) harboring Gln114, ces(340) and CeMA are caused by mutations encoding for Trp114.1,2 The model presented by Westhoff et al.1 suggests conformational changes due to Trp114 that contribute to the expression pattern of the JAL antigen. The weak JAL expression in Rhce(R114Q) may be due to the different biochemical properties of Trp (W) and Gln (Q). However, the JAL expression in ces(340), which also harbors Trp114, is distinctly weaker than in CeMA and, hence, requires a different molecular explanation (Table S3, available online):1,2,8 The additional Leu245Val substitution, known to cause a weak expression of the e antigen,8 could also induce the weak expression of the JAL antigen. Currently observed alleles cannot exclude the possibility that the weak JAL expression in both Rhce variants is caused by the 4 amino acid residues at positions 48, 60, 68, and 103, which typically differ between the RhCe and Rhce proteins. We conclude that JAL and JAHK antigens are expressed by Ce and ce variants of the RhCE protein. We found that neither antigen was expressed by any of the 4 RhD protein variants possessing similar amino acid substitutions at positions 114 and 122.