A transfection model for weak rhesus D expression

Abstract

Weak D is recently reported to be encoded by 14 different weak D genotypes. The amount of RHD mRNA in donors expressing weak D was measured in previous studies. By semiquantitative RT‐PCR analysis Rouillac et al. (Blood 1996) found lower RHD mRNA levels in donors expressing weak D compared to normal RhD donors, whereas Beckers et al. (Transfusion 1997) described equal mRNA levels. The aim of the present study is to explore whether the pointmutations found in donors carrying the weak D phenotype can be responsible for weak D expression. Real‐time quantitative PCR analysis showed that levels of RHD mRNA from donors expressing different weak D genotypes (type 1, 2 and 3) did not differ from RHD mRNA levels from normal heterozygous RhD donors. This indicates that the mutations found in weak D genotypes do not affect RhD expression on transcription level. To investigate whether the described mutations influence translation of mRNA into RhD protein or the configuration of the RhD protein, weak D type 1 cDNA (T809G) and weak D type 3 cDNA (C8G) was transfected in K562 cells. Preliminary results indicate that the ratios between RhD expression and RHD mRNA level (RhD expression/RHD mRNA level) do not differ between weak D type 1 and normal D K562 cells, but are reduced in weak D type 3 transfectants compared to normal D K562 cells. This suggests that in K562 neither the translation nor the configuration of RhD is influenced by the mutation T809G. There might be a missing or limited factor in the K562 model involved in the expression of RhD, hampering the interpretation of the weak D type 1. However, the lower expression of RhD with the mutation C8G might be caused by changed configuration or changed intracellular transport to the membrane.

Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety

“…When serum from sample 5 was adsorbed on RBCs from sample 3 and then eluted, the reactivity was anti-e. Variants 1 to 5 exhibited the specific profile for the DAR partial phenotype when tested with a panel of anti-D MoAbs (data not shown). 25 This result was consistent with the anti-D found in sera 2 to 4.…”

“…Known transcripts as well as new transcripts were identified (Table 4, Figure 1). For individual 1, 3 different transcripts were found: (1) the DAR allele, 25 carrying 602CϾG (Thr201Arg) in exon 4, 667TϾG (Phe223Val) in exon 5, and 1025TϾC (Ile342Thr) in exon 7; (2) the ceAR allele 25 carrying 48GϾC (Trp16Cys) in exon 1; 712AϾG (Met238Val), 733CϾG (Leu245Val), 787AϾG (Arg263Gly), and 800TϾA (Met267Lys) in exon 5 and 916AϾG (Ile306Val) in exon 6; (3) the new ceEK allele, carrying 48GϾC (Trp16Cys) in exon 1; …”