Molecular basis of RhD-negative phenotype in North Indian blood donor population

Abstract

Background & objectives: RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population.

Methods: Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHDΨ) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system).

Results: No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHDΨ gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHDΨ gene.

Interpretation & conclusions: RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHDΨ gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

Keywords: Blood donor; RH blood group; RHD; RhD negative; gene; genetic basis; genotype; phenotype; polymorphism.

Fig. 1

PCR-sequence specific priming products for intron 4 region of RHD gene (n=200). D1-D5 shows reaction of five donor samples using primer set A1-A2, Ctrl-control sample (DNA from known RhD-positive individual was used as control), L₅₀ - 50 bp DNA ladder. Band at 1200 bp is a product of RHCE gene while band 600 bp is the product from intron region of RHD gene. None of the five samples shown in the image carried intron 4 region of the RHD gene while both the bands were observed in the control sample.

Fig. 2

PCR-sequence specific priming products for exon 10 region and normal / RHDΨ segment in exon 4 region of RHD gene (n=200). L₁₀₀ - 100 bp DNA ladder, L₅₀ - 50 bp DNA ladder. Reaction with primer set B1-B2. (A) shows a 193 bp product in the control sample (Ctrl) and no product in donor sample (D61). Reaction with primer set C1-C2. (B) shows a 381 bp product obtained in control sample and no product in donor (D105) sample. Presence of pseudo gene (RHDΨ) would have resulted in a 418 bp product instead of 381 bp product seen in sample with normal RHD gene.

Fig. 3

PCR-sequence specific priming and gel electrophoresis for presence of RHD gene in an individual with RhD-negative phenotype. L₁₀₀ - 100 bp DNA ladder, L₅₀ - 50 bp DNA ladder. Reaction of study samples (D11, D12, D14) with all three primer sets used in the study are shown here. D14 sample amplification resulted in all three products expected from a normal RHD gene as in the control sample (Ctrl) from normal RhD-positive individuals. Only 1200 bp products were observed for sample D12 representing amplification of region in the homologous portion of RHCE gene.