|Year : 2022 | Volume
| 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
|Date of Submission||18-Feb-2022|
|Date of Decision||01-Mar-2022|
|Date of Acceptance||03-Mar-2022|
|Date of Web Publication||29-Apr-2022|
Dr. Mujtaba Ali Allawati
Department of Medical laboratory Science, Oman College of Health Sciences, Wattyah
Source of Support: None, Conflict of Interest: None
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.
Keywords: DBS-0, Oman, RHD gene
|How to cite this article:|
Allawati MA, Balushi BA. A possible novel DBS-0 like allele. Glob J Transfus Med 2022;7:77-9
| 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. 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. The immunogenicity of Rh antigens differs, with D antigen being the most immunogenic. 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 as seen in whites or RHDψ gene or hybrid RHD-CE-Ds gene 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. 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. Moussa et al.'s study realized that a partial D sample type DBT was mistyped as D negative by serological tests. 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. The frequency of D negative in Omanis is 8.35% 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.
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. 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. Exon 5 of the hybrid composed of RHCE (667–712) and RHD that gives four amino acids substitutions in the protein [Figure 1]. 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: Molecular bases of DBS-0 allele with hybrid RHD-RHCE/D-RHD. The black boxes represent wild-type RHD exons 1 through 10. The white box represents the RHCE within exon 5. The presence of RHCE within RHD gene in part of exon 5 area causes (F223V), (A226P), (E233Q), and (V238M) amino acid substitutions. A226P substitution (marked red) is responsible for the amplification of RhE allele|
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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: Serological Rh phenotyping of the sample. The gel card analysis showed D − C + c + E − e+ phenotype (a). D negative was confirmed by weak D testing of the sample which was performed along with direct anti-human globulin test as well to rule out any false-positive weak D result (b)|
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|Figure 3: Molecular analysis of Rh alleles for the sample. BAGene RH-TYPE gel view shows self-explanatory lanes for each RH allele. Lane M denotes for 100 bp DNA ladder. A polymerase chain reaction positive control with Human Growth Hormone that gives a 434 bp was run along each polymerase chain reaction for the detection of RH allele. The polymerase chain reaction gel view showed 162 bp for RhC allele, 145 bp for Rhc allele, 157 bp for RhE allele, and 155 bp for Rhe allele|
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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: DBS-0 like allele in cis to DAR2.00 allele. BAGene Partial D-TYPE gel view showed lanes 1-10 for RHD exons 1-10, respectively. Lane M is FastRuler™ LowRange DNA Ladder. This showed amplification of exon 1 (134 bp), 2 (146 bp), 3 (118 bp), 4 (135 bp), 6 (132 bp), 7 (120 bp), exon 9 (184 bp), and exon 10 (132 bp). No amplification bands of exon 5 (132 bp) and intron 7/exon 8 (637 bp) in lanes 5 and 8 suggest DBS-0 and DAR2.00, respectively. Since no serological E antigen was detected [Figure 1], the case was considered DBS-0 like allele. A polymerase chain reaction positive control with human growth hormone that gives a 434 bp was run along each polymerase chain reaction. The D status was reclassified as D positive (Partial D)|
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|Figure 5: Schematic diagram on RHD molecular background of the sample. The 10 exons of the RHD (black box) and RHCE (white box) genes are symbolized by squares and numbered 1-10. The white box within RHD box indicates hybrid RHcE involved in DBS-0 partial D allele. The blue line out of RHD exon box denotes for missense mutation involved in DAR 2.00. The sample genotype possibly composed of the variant DBS-0 in cis to DAR 2.00 (DCe) haplotype (top panel) and complete RHD gene deletion (bottom panel). A possible novel DBS-0 like allele is composed of hybrid RHcE (226P) (5:667-5:712) in RHD (top panel). The presence of RHcE (226P) is responsible for the detection of RhE allele|
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|Figure 6: Singleplex real-time polymerase chain reaction analysis for the detection of RHD exon 5. QuantiNova SYBR®Green real-time polymerase chain reaction was performed using Rotor-Gene Q. Both Rh-positive control and the sample 82 (case report sample) showed amplification of RHD exon 5 (a). To confirm the correct exon was amplified, DNA gel electrophoresis was performed and showed 157 bp amplification of the desired RHD exon 5 with Rh+ (Bu and NE) controls and sample 82 (b). Lane C is FastRuler™ Low Range DNA Ladder|
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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. 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.,, 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.
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
Conflicts of interest
There are no conflicts of interest.
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