|Abstract||Aberrant RHD alleles are much more frequent in Africans than in Europeans. A comprehensive survey of the variability of RHD alleles among D positive Europeans at the molecular level was lacking. Systematic nucleotide sequencing of any RHD exon was not done before in random donors. We performed a random survey among blood donors in South-Western Germany: 500 samples of Rhesus phenotype ccDee, 250 CcDee and 250 ccDEe. They were tested by PCR-SSP for selected nucleotide variations that are coding for S3C (indicative of weak D type 3); L110P (DVII); A149D (weak D type 5); N152T; T201R and F223V (weak D type 4); V270G (weak D type 1); T283I (DHMi); M295I (weak D type 11); G355S (DNB); T379M (DAU-0); and G385A (weak D type 2). All PCR-SSP positive samples were confirmed by nucleotide sequencing of the ten RHD exons from genomic DNA. The nucleotide sequence for RHD exon 5 was tested in all 1000 samples.
In the ccDee phenotype, 5 samples were positive for weak D type 4.0; 3 samples for weak D type 4.1; 5 samples for DAU-0; 1 sample for weak D type 11; 1 sample for DV type 1 + DAU; the molecular basis of the latter sample was determined as RHD(F223V, E233Q, T379M) and dubbed DAU-5. There were 2 DNB in the CcDee samples. No variant RHD allele was found in the ccDEe samples. We determined the population frequency of the detected alleles. Nucleotide sequencing RHD exon 5 revealed three new alleles RHD(G212G), RHD(R234W) and RHD(V245L).
There are four known D clusters within the RHD phylogeny: DIVa cluster, weak D type 4 cluster, DAU cluster and Eurasian D cluster. Within the phylogeny of the RHD alleles, there are two known recombination events between the clusters. The recombination between the DIVa cluster and weak D type 4 cluster is represented by the DIII type 5 allele. The recombination between the DIVa cluster and Eurasian D cluster is represented by the DIII type 4 allele. In this study, we found the DAU-5 allele, which represents another example for an allele that was formed by a recombination event. In this case, the DAU-5 represents a recombination between the DAU cluster and Eurasian D cluster. The specific allele of the Eurasian D cluster that is involved in this recombination event was DV type 1.
In conclusion, we found 17 variant RHD alleles by a PCR-SSP screen, which are consistent with previous reports. The novel allele RHD(F223V, E233Q, T379M) was dubbed DAU-5 and represented a recombination between two known alleles of the DAU cluster and Eurasian D cluster. By sequencing RHD exon 5 three novel alleles were found. In such a limited screen, we characterized 4 novel RHD alleles: DAU-5, RHD(G212G), RHD(R234W) and RHD(V245L). DAU-5 added to the understanding of the RHD phylogeny. Our approach showed that - even within the European population - the variety of RHD alleles may be larger than anticipated and biologic relevant information may be gleaned from such systematic studies.||dc.description.abstract