Original Article

Rhesus Box as the Primary Mechanism of RHD Gene Deletion in RhD-Negative Blood Donors from Eastern Iran

Abstract

Background: The Rh blood group system is highly significant in transfusion medicine because of the strong immunogenicity of the D antigen. The RhD-negative phenotype arises through various molecular mechanisms in different populations, most commonly complete deletion of the RHD gene caused by unequal recombination between upstream and downstream Rhesus box sequences. Although this mechanism has been well documented in some populations, limited data are available from Iran, particularly its eastern regions. This study aimed to determine the molecular basis of the RhD-negative phenotype among blood donors in eastern Iran.

Materials and Methods: In this cross-sectional study, a total of 16,190 blood donors referred to blood transfusion centers in South Khorasan Province, eastern Iran, over a one-year period were screened serologically for RhD status. Among them, 2,198 individuals were identified as RhD-negative, and 100 serologically confirmed RhD-negative donors were randomly selected for molecular evaluation. RhD typing was performed using standard serologic methods and verified by indirect antiglobulin testing. Molecular investigations included PCR–sequence-specific priming (PCR-SSP) targeting RHD exons 5, 7, and 10, real-time PCR for confirmation, and PCR–restriction fragment length polymorphism (PCR-RFLP) to detect the hybrid Rhesus box and determine RHD zygosity.

Results: Among 16,190 blood donors screened during the study period, 2,198 (13.57%) were identified as RhD-negative. From this group, 100 samples were randomly selected for molecular analysis. Both PCR-SSP and real-time PCR confirmed the absence of RHD exons 5, 7, and 10 in all samples, indicating complete deletion of the RHD gene. PCR-RFLP analysis further confirmed that all donors were homozygous for the hybrid Rhesus box, with full concordance observed between exon-specific assays and hybrid Rhesus box genotyping.

Conclusion: These findings indicate that the RhD-negative phenotype in eastern Iran is primarily due to homozygous RHD gene deletion mediated by the hybrid Rhesus box. Hybrid Rhesus box analysis may therefore serve as a reliable molecular marker for accurate RhD typing, which could improve transfusion safety and perinatal management in this population.

1. Flegel WA. The genetics of the Rhesus blood group system. Blood transfus. 2007;5(2):50-7.
2. Sassi A, Ouchari M, Houissa B, et al. RHD genotyping and its implication in transfusion practice. Transfus Apher Sci. 2014;51(3):59–63.
3. Gammon RR, Conceicao M, Benitez N, et al. Comparison of RhD typing results by serology and molecular methods. Lab Med. 2023;54(2):190–2.
4. Yazer MH, Panko G, Holcomb JB, et al. Not as “D” eadly as once thought–the risk of D-alloimmunization and hemolytic disease of the fetus and newborn following RhD-positive transfusion in trauma. Hematology. 2023;28(1):2161215.
5. Daniels G. The molecular genetics of blood group polymorphism. Transpl Immunol. 2005;14(3-4):143–53.
6. Clausen FB, Damkjær MB, Dziegiel MH. Noninvasive fetal RhD genotyping. Transfus Apher Sci. 2014;50(2):154–62.
7. Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011;44(1):81–91.
8. Fung M, Eder A, Spitalnik S. Technical Manual 19th ed Bethesda. MD AABB. 2017.
9. McGowan EC, Flower RL, O'Brien H, et al. Non-invasive prenatal testing for sub-saharan Africa: Tailoring approaches for foetal RHD genotyping in RHD-negative pregnant women to manage African-associated RHD Alleles. Africa Sanguine. 2017;19(1):18–29.
10. Papasavva T, Martin P, Legler TJ, et al. Prevalence of RhD status and clinical application of non-invasive prenatal determination of fetal RHD in maternal plasma: a 5 year experience in Cyprus. BMC Res Notes. 2016;9:198.
11. Amaral DR, Credidio DC, Pellegrino Jr J, et al. Fetal RHD genotyping by analysis of maternal plasma in a mixed population. J Clin Lab Anal. 2011;25(2):100–4.
12. Moussa H, Tsochandaridis M, Chakroun T, et al. Molecular background of D‐negative phenotype in the Tunisian population. Transfus Med. 2012;22(3):192–8.
13. Rodrigues A, Rios M, Pellegrino Jr J, et al. Presence of the RHD pseudogene and the hybrid RHD-CE-Ds gene in Brazilians with the D-negative phenotype. Braz J Med Biol Res. 2002;35(7):767–73.
14. Sadeghi-Bojd Y, Amirizadeh N, Oodi A. RHD genotyping of Rh-negative and weak D phenotype among blood donors in Southeast Iran. Int J Hematol Oncol Stem Cell Res. 2021;15(4):213-220.
15. Khosroshahi BN, Oodi A, Namjou S, et al. RHD genotyping by molecular analysis of hybrid rhesus box in RhD-negative blood donors from Iran. Indian J Hematol Blood Transfus. 2019;35(1):119-124.
16. Oliveira J, Osório N, Rocha J, et al. Fetal RHD and RHCE genotyping in plasma of Rh negative pregnant women. Int J Biomed Lab Sci (IJBLS). 2012;1(2):50–8.
17. Wagner FF, Flegel WA. RHD gene deletion occurred in the Rhesus box. Blood. 2000;95(12):3662–8.
18. Christensen RD, Bahr TM, Ilstrup SJ, et al. Alloimmune hemolytic disease of the fetus and newborn: genetics, structure, and function of the commonly involved erythrocyte antigens. J Perinatol. 2023;43(12):1459–67.
19. Stegmann TC, Veldhuisen B, Bijman R, et al. Frequency and characterization of known and novel RHD variant alleles in 37 782 Dutch D‐negative pregnant women. Br J Haematol. 2016;173(3):469–79.
20. Sandler SG, Flegel WA, Westhoff CM, et al. It’s time to phase-in RHD genotyping for patients with a serological weak D phenotype. Transfusion. 2015;55(3):680-9.
21. Wagner FF, Gassner C, Müller TH, Schönitzer D, Schunter F, Flegel WA. Molecular Basis of Weak D Phenotypes. Blood. 1999;93(1):385–93.
22. Flegel WA, Denomme GA, Queenan JT, et al. It's time to phase out “serologic weak D phenotype” and resolve D types with RHD genotyping including weak D type 4. Transfusion. 2020;60(4):855–9.
23. Fung MK, Grossman BJ, Hillyer CD, et al. AABB technical manual. Bethesda, MD, USA: AABB. 2014.
24. Westhoff CM. The Rh blood group system in review: a new face for the next decade. Transfusion. 2004;44(11):1663-73.
25. Li Q, Hou L, Guo ZH, et al. Molecular basis of the RHD gene in blood donors with DEL phenotypes in Shanghai. Vox Sang. 2009;97(2):139–46.
26. Al Lawati MA. Molecular background of serological D negative phenotype in the Omani population: Liverpool John Moores University (United Kingdom); 2021.
27. Alalshaikh MA, Alsughayir AH, Alsaif AS, et al. Molecular background of RhD-positive and RhD-negative phenotypes in a Saudi population. Saudi J Med Med Sci. 2024;12(3):210-215.
28. Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011;44(1):81–91.
Files
IssueVol 20 No 2 (2026) QRcode
SectionOriginal Article(s)
Keywords
RhD-negative; RHD gene deletion; Hybrid Rhesus box; PCR-SSP; PCR-RFLP
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Nakhaei Shamahmood M, Zangooie A, Anani Sarab G, Sajjadi SM, Salehi Z, Mezginejad F. Rhesus Box as the Primary Mechanism of RHD Gene Deletion in RhD-Negative Blood Donors from Eastern Iran. Int J Hematol Oncol Stem Cell Res. 2026;20(2):127-134.