Global Journal of Transfusion Medicine

SPECIAL COMMUNICATION
Year
: 2022  |  Volume : 7  |  Issue : 1  |  Page : 77--79

A possible novel DBS-0 like allele


Mujtaba Ali Allawati1, Badria Al Balushi2,  
1 Department of Medical laboratory Science, Oman College of Health Sciences, Wattyah, Oman
2 Central Blood Bank, Muscat, Bausher, Oman

Correspondence Address:
Dr. Mujtaba Ali Allawati
Department of Medical laboratory Science, Oman College of Health Sciences, Wattyah
Oman

Abstract

RHD variants are divided into three categories; weak D, partial D, and Del. The variants detection is important during donor testing and pregnancy. Studies shown that the detection of D variants may be missed by standard serologic methods including Indirect Antihuman Globulin Test and may cause anti-D immunization when exposed to D-negative individual. The limitations of serology can be overcome by RHD gene molecular typing. We describe for the first time a possible novel DBS-0 like allele partial D which revealed serological D and E negative in an Omani donor that is different from the previously described DBS-0 with serological positive D and partial E.



How to cite this article:
Allawati MA, Balushi BA. A possible novel DBS-0 like allele.Glob J Transfus Med 2022;7:77-79


How to cite this URL:
Allawati MA, Balushi BA. A possible novel DBS-0 like allele. Glob J Transfus Med [serial online] 2022 [cited 2022 Dec 8 ];7:77-79
Available from: https://www.gjtmonline.com/text.asp?2022/7/1/77/344329


Full Text



 Introduction



The human Rh blood group system is the most important system clinically after the ABO group system. The Rh blood group system has two main genes: RHD encodes the D antigen and RHCE encodes for C/c and E/e antigens both with 10 exons.[1] RHD and RHCE genes each produce a protein antigen with 417 amino acids long. The most important antigens of the Rh system are D, C. c, E and e.[2] The immunogenicity of Rh antigens differs, with D antigen being the most immunogenic.[3] To prevent alloimmunization due to anti-D, exposure of D-negative individuals to D-positive red blood cells (RBCs) should be avoided. Therefore, correct D phenotyping of donor's RBCs is essential to avoid such anti-D alloimmunization.

An individual can either be D-positive or D-negative. D-positive results from the existence of whole wild type RHD gene, many RHD-CE-D hybrids that give a partial D or a point mutation on RHD gene that raises either weak D or partial D. The absence of D antigen is due to a complete deletion of RHD gene[4] as seen in whites or RHDψ gene[5] or hybrid RHD-CE-Ds gene[6] as seen in African.

In most laboratories, serology is the method of choice for the detection of D antigen; however, it has limitations. Studies shown that the detection of D variants such as weak D, Del phenotype, and partial D may be missed by standard serologic methods including Indirect Antihuman Globulin Test and may cause anti-D immunization when transfused to D-negative recipients. Garratty calculated that at least 120 weak D or Del donors, typed D-negative serologically, are transfused to D-negative recipients annually in Southern California.[7] In another study by Flegel et al. on 46,133 serologically D negative donors, the RHD genotyping showed that 96 samples had RHD gene, half of which harbored Del phenotype.[8] Moussa et al.'s study realized that a partial D sample type DBT was mistyped as D negative by serological tests.[9] The limitations of serology can be overcome by RHD gene molecular typing.

The D-negative phenotype has a high molecular diversity which explains the discrepancies found between serologic and molecular methods.[10] The frequency of D negative in Omanis is 8.35%[11] but the molecular background explaining this phenotype is unknown in this population. In an aim to explore the molecular background of a serological D negative for any D variants possibility, we report a case with the presence of entire RHD gene in an Omani blood donor. We describe a possible novel DBS-0 like allele which revealed serological D and E negative that is different from the previously described DBS-0 with serological positive D and partial E.[12]

An A Rh (D) negative Omani male donor passed all eligibility criteria tests and donated blood. Serological Rh phenotyping showed D-C+c+E-e+ phenotype giving initial impression of possible dCe/dce genotype. Molecular analysis for RHD exons 1 through 10 using BAGene Partial D-TYPE (BAG Health Care, Germany) revealed the amplification of all RHD exons with the exception of exons 5 and 8. Singleplex real-time polymerase chain reaction (RT-PCR) for RHD exons (RHD exon 8 was not tested) revealed the presence of all exons. BAGene RH-TYPE (BAG Health Care, Germany) showed amplification of RHC, RHE, RHc, and RHe alleles. The combination of serological Rh phenotyping, BAGene, and RT-PCR results suggest the existence of possible novel DBS-0 like allele in cis to DAR 2.00. The serological D negative was considered a false D-negative with a possible DCe/dce genotype.

 Discussion



RHD variants are divided into three categories; weak D, partial D, and Del.[13] DV Type III (DBS-0) first described in 1996 is a type of partial D that is characterized by the existence of partial D and E antigens with DCe haplotype. The partial E expression on RBCs is as a result of hybrid Rh gene that is composed of RHD (exons 1-4)-RHCE/RHD (exon 5)-RHD (exons 6-10), as no Rh E transcript was found by RT-PCR.[12] Exon 5 of the hybrid composed of RHCE (667–712) and RHD that gives four amino acids substitutions in the protein [Figure 1].[14] We report for the first time a possible novel case of DBS-0 like partial D with serological D negative in an Omani donor.{Figure 1}

The serological Rh phenotyping of the donor revealed D−C+ c+E−e+ [Figure 2]; however, molecular analysis showed the amplification of RHC, RHE, RHc, and RHe alleles [Figure 3]. Incompatibility between serological E negative and the existence of RhE allele in molecular analysis prompted to do further molecular analysis.{Figure 2}{Figure 3}

We performed BAGene Partial D-TYPE conventional PCR testing which showed amplification of all RHD exons with exception of exon 5 and 8 [Figure 4]. This suggested hemizygous DBS-0 in cis to DAR allele/del [Figure 5]. Singleplex RT-PCR for the amplification of RHD exons 1 through 10 (except exon 8) on the sample was also performed which showed amplification of all exons. The discrepancy in RHD exon 5 amplification between singleplex RT-PCR and BAGene Partial D-TYPE was due to the primer pair used for each test. Singleplex RT-PCR used sequence-specific primer (SSP) that targets nucleotide 654 on RHD exon 5 and thus amplified the exon successfully [Figure 6]. In contrast, BAGene SSP targets one of the nucleotides that has been mutated due to the RHD-RHCE-RHD hybrid gene existence and thus failed to amplify RHD exon 5.{Figure 4}{Figure 5}{Figure 6}

Nucleotide's substitutions in RHCE give Rh C/c and Rh E/e polymorphisms. A single nucleotide substitution (P226A) is responsible for the expression of E to e polymorphism.[14] We assume the amplification of RhE allele seen in BAGene RH-TYPE is due to the amino acid substitution (A226P) in exon 5 of the hybrid RHD-RHCE/D/RHD that is specific for RhE allele. However, mRNA transcript should be studied to rule out the existence of Rh E. The amplification of RhE allele due to its existence in the hybrid RHD-RHCE/D-RHD corresponds to the previously described DBS-0 partial D case, and hence we named it DBS-0 like allele (haplotype DCe).

RBCs with D variants such as partial D may cause serological discrepancy in D antigen detection. These discrepancies are mainly due to the use of different marketed anti-D reagents.[15],[16],[17] For weak D testing, we used BioRAD ID-DiaClon anti-D which failed to detect the novel DBS-0 like partial D and explains the false serological D-negative result. This drives us to realize the importance of studying the molecular background of D negative to avoid D mistype in donors and prevent possible anti-D alloimmunization.

 Conclusion



We report for the first time a possible novel case of DBS-0 like allele with DCe haplotype giving a partial D phenotype in an Omani donor. Our observation drives us to realize the necessity to study the molecular background of D negative in this population. Molecular analysis of the transcript would be a good approach to further explore this case.

Acknowledgment

We would like to thank the Ministry of Health Oman for sponsoring the research (as part of PhD research). We would also like to thank Dr. Zainab Al Araimi, Director of Central Blood Bank Oman for the continuous support during the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Chérif-Zahar B, Mattéi MG, Le Van Kim C, Bailly P, Cartron JP, Colin Y. Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. Hum Genet 1991;86:398-400.
2Flegel WA. The genetics of the Rhesus blood group system. Blood Transfus 2007;5:50-7.
3Pittiglio DH. Modern Blood Banking and Transfusion Practices. Philadelphia: Davis; 2012.
4Colin Y, Chérif-Zahar B, Le Van Kim C, Raynal V, Van Huffel V, Cartron JP. Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 1991;78:2747-52.
5Singleton BK, Green CA, Avent ND, Martin PG, Smart E, Daka A, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood 2000;95:12-8.
6Faas BH, Beckers EA, Wildoer P, Ligthart PC, Overbeeke MA, Zondervan HA, et al. Molecular background of VS and weak C expression in blacks. Transfusion 1997;37:38-44.
7Testing for weak D. Vox Sang 2006;90:140-53. Available from: https://pubmed.ncbi.nlm.nih.gov/16523560/. doi: 10.1111/j.1423-0410.2006.00717.x. [Last accessed on 2022 Mar 26].
8Flegel WA, von Zabern I, Wagner FF. Six years' experience performing RHD genotyping to confirm D- red blood cell units in Germany for preventing anti-D immunizations. Transfusion 2009;49:465-71.
9Moussa H, Tsochandaridis M, Chakroun T, Jridi S, Abdelneji B, Hmida S, et al. Molecular background of D-negative phenotype in the Tunisian population. Transfus Med 2012;22:192-8.
10Sassi A, Ouchari M, Houissa B, Romdhane H, Abdelkefi S, Chakroun T, et al. RHD genotyping and its implication in transfusion practice. Transfus Apher Sci 2014;51:59-63.
11Allawati M, AlKalbani L, Al Balushi T. ABO and Rh (D) Phenotypes, Allele Frequencies and Estimated Genotypes in Omanis: A retrospective study from armed forces hospital. JMSCR 2021;9:1-7.
12Avent ND, Finning KM, Liu W, Scott ML. Molecular biology of partial D phenotypes. Transfus Clin Biol 1996;3:511-6.
13Flegel WA, Wagner FF. Molecular biology of partial D and weak D: Implications for blood bank practice. Clin Lab 2002;48:53-9.
14Avent ND, Reid ME. The Rh blood group system: A review. Blood 2000;95:375-87.
15Kulkarni SS, Vasantha K, Gupte SC, Mohanty D, Ghosh K. Potential of commercial anti-D reagents in the identification of partial D variants in Indian population. Indian J Med Res 2007;125:641-4.
16Wang D, Lane C, Quillen K. Prevalence of RhD variants, confirmed by molecular genotyping, in a multiethnic prenatal population. Am J Clin Pathol 2010;134:438-42.
17Kabiri Z, Benajiba M, Hajjout K, Dakka N, Bellaoui H. Testing for partial RhD with a D-screen diagast kit in moroccan blood donors with weak D expression. J Blood Transfus 2014;2014:204301.