RHD Genotyping by Molecular Analysis of Hybrid Rhesus box in RhD-Negative Blood Donors from Iran

Abstract

D antigen is the most important and immunogenic antigen of the Rh blood group. The RhD-negative phenotype has different genetic backgrounds with variable distribution in different populations. Hybrid Rhesus box , resulting from RHD gene deletion, is used in genotyping studies of the Rh blood group as a marker to identify the RHD gene deletion. This study for the first time identified genetic mechanisms for the occurrence of RhD-negative phenotype among the Iranian population. 200 RhD-negative blood donors were randomly selected from Tehran Blood Transfusion Center. The phenotype of D, C, Ε, e and c antigens was serologically identified, and DNA was extracted from buffy coat. The molecular analysis of hybrid Rhesus box was performed by PCR-SSP and PCR-RFLP. Moreover, the presence of different exons of RHD gene was investigated by real-time PCR on extracted DNA. Hybrid Rhesus box was detected in all samples, and PCR-RFLP confirmed that 198 (99%) were homozygous for an RHD gene deletion and 2 were heterozygous for hybrid Rhesus box in which one (0.5%) had a weak D type 11 and the other one (0.5%) had a RHD - CE (2 - 9) - D 2 hybrid allele. Similar to Caucasians, the frequency of RHD gene deletion was high among the Iranian population studied in this investigation, so hybrid Rhesus box can be used as an efficient marker to detect RHD gene deletion in our population.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma

“…Knowing the genetic background of RHD in the population under study would be probably helpful in such cases. A study conducted by Khosroshahi et al in 2019 in Iran on 200 RHD negative individuals showed that 99% of them were homozygous for the complete deletion of the RHD gene, and the remaining 1% could not be identified with the serological reagents used in that study due to weak and partial RHD variants ( 23 ). Also, in another study conducted in Iran in 2021 on 200 subjects, 99% of the RHD negative cases had the mechanism of homozygous deletion of the RHD gene and nonfunctional variants were observed in 1% of cases ( 24 ).…”

RHD Genotyping of Rh-Negative and Weak D Phenotype among Blood Donors in Southeast Iran

“…They showed that 98% of the donors had a hybrid Rhesus box genomic fragment in both RH gene alleles. Two percent of the donors had a non-deletion RHD gene allele by PCR-RFLP 18 . Which is consistent with the present study; therefore, the genetic mechanism of the Rh-negative phenotype in Sistan and Baluchestan ethnic groups in Iran is also due to forming a hybrid Rhesus box.…”

A novel RHD allele caused by RHD c.710_713dup associated with a negative phenotype in a Chinese Han blood donor

“…The RHD gene comprise 10 exons, and most D-negative individuals lack all exons of the RHD gene. [1][2][3] This report describes a novel allele caused by RHD c.710_713dup in a Chinese Han blood donor with D-negative phenotype. S1.…”

“…About 90 D‐negative alleles involving various mutational mechanisms have been identified so far, according to ISBT allele nomenclature version v5.0 (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-groug-terminology). The RHD gene comprise 10 exons, and most D‐negative individuals lack all exons of the RHD gene 1–3 …”