Fiber-FISH Analysis Revealed Genomic Variations in the RHD as a Cause of Serologically Rh-negative Phenotype

Abstract

The human Rhesus (Rh) blood group is determined by 2 highly homologous RH genes, RHD and RHCE. These genes are located on chromosome 1p36.1, a genomic region representing copy number variation. In Rh-positive individuals, both RHD and RHCE genes are present, while in most Rh-negative individuals RHD genes are missing and are homozygous for a single RHCE gene. Some individuals with a serologically Rh-negative phenotype were diagnosed to be RHD-gene positive by a locus-specific DNA assay using polymerase chain reactions with sequence-specific primers (PCR-SSP). Therefore, the current PCR-based assay, examining the presence or absence of partial sequences of RHD genes, does not necessarily produce correct typing results. Alterations in the genomic structure of RHD that cause negative gene expressions are difficult to detect by the PCR-based assay because of highly homologous sequences of the 2 RH genes. In the present study, we examined, by extended chromatin fiber fluorescence in situ hybridization (fiber-FISH) with 2 DNA probes for introns 3 and 7 of the RH genes, the genomic structure of the RH gene region in 6 Rh-negative Japanese donors whose genotype was diagnosed as RHD-gene positive by the PCR-SSP assay. We demonstrated that varied sizes of deletion and/or insertion in the RH loci caused the Rh-negative phenotype.