RHD-Positive Alleles among D- C/E+ Individuals from India

Abstract

Background: Molecular bases of blood group systems, including Rh blood group, have been poorly studied in the Indian population so far, while specificities of Europeans, East Asians and Africans have been well known for years. In order to gain insights into the molecular bases of this population, we sought to characterize the RHD allele in D- Indian donors expressing C and/or E antigen(s).

Methods: RHD gene was analyzed in 171 serologically D-, C/E+ samples by standard molecular methods such as quantitative, multiplex PCR of short fluorescent fragments (QMPSF) and direct sequencing when necessary.

Results: RHD whole gene deletion at the homozygous state was found to be the most common genotype associated with D- phenotype (118/171, 69.0%). Nonfunctional, negative hybrid genes with reported molecular backgrounds were observed in approximately one-third of the samples, while only four samples carry single-nucleotide variations, including one novel nonsense (RHD(Y243X)), one novel frameshift (RHD(c.701delG)), and two missense (RHD(T148R) and RHD(T148R, T195M)) alleles.

Conclusion: Overall we report for the first time the molecular bases of D antigen negativity in the D-, C/E+ Indian population, which appears to be qualitatively similar to other populations, but with a population-specific, quantitative distribution of D-- alleles.

Keywords: Allele; D-negative; Molecular basis; Phenotype; RHD.

Fig. 1

Genotyping D-, C/E+ samples by RHD QMPSF analysis. Typical profiles obtained in samples with A wild-type RHD (hemizygous calibrator sample, Ccee); B homozygous RHD deletion (ccEe); C homozygous RHD deletion (Ccee); D hemizygous RHD-CE(3–8)-D (Ccee); E hemizygous RHD-CE(4–9)-D (Ccee); and F hemizygous RHD-CE(3–9)-D (Ccee). *Absence of the respective exon markers; HFE and F9: positive control gene markers; e1 to e10: RHD exons 1 to 10; x-axis: base pairs; y-axis: relative fluorescence intensity.

Fig. 2

Molecular analysis of the RHD locus in D-, C/E+ samples. A Genotyping data of the compound heterozygous sample by RHD QMPSF analysis (left, RHD-CE(4–9)-D) and Sanger sequencing (right, RHD(c.701delG)). Sanger sequencing profiles in samples presenting with the novel BRHD(T148R), CRHD(Y243X), and DRHD(T148R, T195M) alleles. *Copy number variation of RHD exon markers (i.e. n = 1, while two copies of the gene were calculated); HFE and F9: positive control gene markers; e1 to e10: exons 1 to 10; arrowhead: position of single nucleotide variation.