Jing Wang, Changyan Li, Lian Duan, Wenjun Que, Yan Xing, nTan Bi, Tingxi Zhan, Zebo Yu. RhCE-D (2)-CE hybrid allele causing puzzle in serology and sequencing: a case report[J]. Blood&Genomics, 2020, 4(2): 151-155. DOI: 10.46701/BG.2020022020113
Citation:
Jing Wang, Changyan Li, Lian Duan, Wenjun Que, Yan Xing, nTan Bi, Tingxi Zhan, Zebo Yu. RhCE-D (2)-CE hybrid allele causing puzzle in serology and sequencing: a case report[J]. Blood&Genomics, 2020, 4(2): 151-155. DOI: 10.46701/BG.2020022020113
Jing Wang, Changyan Li, Lian Duan, Wenjun Que, Yan Xing, nTan Bi, Tingxi Zhan, Zebo Yu. RhCE-D (2)-CE hybrid allele causing puzzle in serology and sequencing: a case report[J]. Blood&Genomics, 2020, 4(2): 151-155. DOI: 10.46701/BG.2020022020113
Citation:
Jing Wang, Changyan Li, Lian Duan, Wenjun Que, Yan Xing, nTan Bi, Tingxi Zhan, Zebo Yu. RhCE-D (2)-CE hybrid allele causing puzzle in serology and sequencing: a case report[J]. Blood&Genomics, 2020, 4(2): 151-155. DOI: 10.46701/BG.2020022020113
Zebo Yu, MD, Department of Blood Transfusion, the First Affiliate Hospital of Chongqing Medical University, Chongqing 400016, China. Tel: +86-23-89011038, E-mail: yuzebo2001@163.com
The Rh locus, one of the important blood group systems in transfusion medicine, is controlled by three highly homologous genes: RHAG, RHD and RHCE. RHD and RHCE genes both contain 10 exons with opposite orientation, with genetic homology of higher than 92%. Based on this arrangement and configuration, a hybridization variant easily occurs, which causes variant or weak antigen expression. A 46-year-old woman of group A was admitted to hospital with bloody stool. Her RhD phenotype was confusing (agglutination < 1+) as detected by gel card. The unexpected antibody was identified to be anti-D. Only exon 2 of RHD was detected by sequence-speci?c primer polymerase chain reaction (SSP-PCR) with hybrid heterozygosis of c.150T > C, c.178A > C, c.201G > A, and c.307T > C by sequencing. The genotype of RHCE was confirmed to be Ccee by SSP-PCR and the serologic phenotype was Ccee too. However, the sequencing of RhCE was confirmed to be ccee with c.178CC, c.203AA, c.307CC on exon 2. Further analysis found that the difference between them depended on the replacement of exon 2 from RhD. The genotype of this patient was found to be RhCE-D(2)-CE/RhCE, leading to a confusing RhD phenotype.
Following the ABO group, the Rh (RH) locus is the secondary blood group system in transfusion medicine. It is genetically controlled by three highly homologous genes: RHAG, RHD and RHCE. Functionally, RHD encodes the D antigen while RHCE encodes the Cc/Ee antigens[1-2]. People are classed as Rh-positive and Rh-negative depending on the presence or absence of D antigen which is expressed on the surface of cells. Meanwhile, C/c and E/e antigens are associated with incompatible hemolytic transfusion reactions and hemolytic disease of the newborn (HDN)[3]. As to the genetic structure, each one of them consists of 10 exons with a similar exon-intron organization, and have opposing orientation, facing each other with their 3' ends[4]. They are separated by TMEM50A gene (previously called SMP1 for Small Membrane Protein 1), and the 3' and 5' RHD ends are flanked by two homologous regions of DNA called Rhesus boxes[5]. However, the homologous base sequences of RHD and RHCE can be up to 97%[6]. Based on this unique structure, multiple variations occur between them, especially gene conversion of the exons[7]. The hybrid between them can cause D-negative, partial D, Del (weak D that depends on single nucleotide polymorphisms) and so on[4, 8-12]. Here we report a case with RHCE exon 2 partly replaced by RHD exon 2.
2.
CASE REPORT
A 46-year-old woman group A, with a history of systemic lupus erythematosus (SLE) was admitted to hospital with bloody stools. During her hospital stay, pre-transfusion examinations were done by gel card method (Jiangsu Libo Medicine Biotechnology Co. Ltd, China). At first, reverse typing could not be done as the cells were all RhD-positive. The reverse typing was reliably confirmed after the cells were replaced with RhD-negative cells. Alarmingly, the results showed group A, RhD (agglutination < 1+) by gel card, which differed from historical RhD-positive A type blood (detected in another hospital before). However, the RhD remained weak agglutination even after repeated sampling. Monoclonal anti-D (Millipore, USA) was used to confirm the RhD phenotype by antiglobulin card. Unfortunately, the RhD confirmatory test couldn't be done, as the result of direct antiglobulin test (DAT) was positive irrespective of whether the cells were washed or eluted at 56°C for 8 minutes with normal saline. The indirect antiglobulin test(IAT) was positive (4+) and turned to be negative after using group O, RhD-negative cells. What's more, the unexpected antibody was confirmed to be anti-D by panel screening cells (Sanquin, Netherlands) (Table 1). To elucidate the confusing serology results, sequence-specific primer polymerase chain reaction (SSP-PCR) and Sanger sequencing were performed in turn (Jiangsu ZoJiWat Biomedical Co. Ltd, China). This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and informed consent was obtained from the patient.
Table
1.
Serologic results of propositus
Forward typing1
Reverse typing
DAT
IAT3
Unexpected antibody
RhCE phenotype
Anti-A
Anti-B
Anti-D
Ac2
Bc2
Oc2
Washed
Eluted
4+
0
< 1+
0
4+
0
3+
1+
4+
Anti-D
CcEe
The serologic results of propositus were all the same no matter using the primary or secondary blood sample. 1: The self-control well is negative. 2: Cells were all RhD-negative. 3: The IAT was negative after the cells were replaced with RhD-negative.
Firstly, the genomic DNA was extracted, and exons 1 to 10 of RhD were tested by SSP-PCR. The result showed that only exon 2 could be deter-mined (Fig. 1). After sequencing, the result showed that there was a hybrid of RhD and RhCE in exon 2. The hybrid heterozygosis were included as c.150T > C, c.178A > C, c.201G > A, c.203G > A, and c.307T > C (Fig. 2).
Figure
1.
SSP-PCR detection on exons 1 to 10 of RhD.
Each well of SSP-PCR contains primers of control gene and relevant exons. The Tm ranges of them were 76-78 ℃ and 86-88 ℃ respectively. Only exon 2 of RhD was detected while others were negative.
After amplicon from the exon 2 of RHD by standard PCR assay, 5 hybrid heterozygosis were detected by Sanger sequencing, including c.150T > C, c.178A > C, c.201G > A, c.203G > A and c.307T > C.
To further study the concrete mechanism of RhD, the RhCE gene was tested again by SSP-PCR. Specific fragments corresponding to antigen C/c and e were all positive, which hinted that the genotype of RhCE was Ccee (Fig. 3A). To straighten out the relation between RhD and RhCE genes, DNA sequencing was carried out again on gene RhCE. All 10 exons could be detected while there was still some homozygosis located on exons, such as c.48CC on exon 1, c.178CC, c.203AA, c.307CC on exon 2, and c.676GG on exon 5 (Fig. 3B).
Figure
3.
SSP-PCR and Sanger sequencing results of RhCE.
A: SSP-PCR results of RhCE. B: Sanger sequencing results of RhCE. The SSP-PCR verified that the genotype of RhCE was Ccee. However, the key bases in c.48 on exon 1, c.178, c.203 and c.307 on exon 2, and c.676 on exon 5 revealed that the genotype should be ccee.
The Rh system is one of the most important and complex blood groups in transfusion medicine after ABO, due to its high antigenicity[13]. What's more, more than 50 different Rh antigens have been identified[1]. Among these antigens, D and Cc/Ee are of crucial significance for the generation of unex-pected antibodies, especially for anti-D and anti-E, which are responsible for HDN and crossing matches, respectively.
In this case, the patient was confirmed as group A, while her RhD phenotype was inexplicit. As the DAT was positive regardless of the cells being washed or eluted, the RhD confirmatory test couldn't be applied. To further investigate, SSP-PCR was performed for RhD. However, only exon 2 could be determined. Furthermore, the DNA sequencing confirmed that the heterozygosis, such as c.150T > C, c.178A > C, c.201G > A and c.307T > C, existed in exon 2, hinting a hybrid occurred between RhD and RhCE. One of RhD alleles was missing while other was RhCE-RHD(2)-RHCE[14].
SSP-PCR analysis of the RhCE revealed that the RHCE genotype was Ccee, and the serologic phenotype was also Ccee. However, the sequencing of RhCE confirmed to be ccee. The difference between them depended on the replacement of exon 2 from RhD. Based on the sequencing results of c.150CC, c.178CC, c.203AA, c.308CC on exon 2 of RHCE, it was confirmed to be allele RHce, while c.48CC on exon 1 was further confirmed to be allele RHce 48C (BGMUT ID815). In fact, in addition to the existence of exon 2 of RhD with 150T, 178A, 203G, 307T, it also contains the RHCe allele. However, due to the high amplification efficiency of specific primers for exon 2 of normal RhCE, the exon 2 of RhD could not be amplified by PCR and detected by Sanger sequencing. Above all, we supposed that the existence of exon 2 of RhD disturbed the sequencing of RHCE. Recently, an article reported that the presence of RHCE-D5-CE hybrid alleles may cause false-negative DNA-typing results for the Rh e antigen although express the Rh e antigen[9]. Similarly, in this case Rh C antigen was confirmed by sero-typing and SSP-PCR, while tested negative by DNA typing.
As to the phenotype of RhD, partial protein en-coded by the exon 2 of RhD was located outside the cell membrane, which explained why the agglutination of anti-D in the forward typing was not negative. However, it's still confusing why the RhD phenotype was confirmed to be positive in the previous hospital, when upon receiving RhD-positive blood, leading to the production of anti-D. Therefore, it is essential to take effective measures to strengthen blood group typing skills or techniques in primary hospitals.
Above all, the genotype of this patient is RhCE-D(2)-CE/RhCE (Fig. 4), leading to a confusing RhD phenotype. As a patient, it's best to receive RhD-negative blood. However, it should be treated as RhD-positive if the blood was donated (anti-D should be taken into account).
Figure
4.
Structure of RhCE-D(2)-CE/RhCE hybrid alleles.
A: The exon of RhCE was exchanged by the exon 2 of RhD. B: The normal allele of RhCE.
This work was supported by grants from the National Natural Science Foundation of China (No. NSFC81802162) and Chongqing Science and Tech-nology Commission (No. cstc2017jcyjAX0262). The genotyping and Sanger sequencing techniques were supported by Jiangsu ZoJiWat Biomedical Co., Ltd. China.
Conflict of interest: The authors have no conflict of interest to report.
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The serologic results of propositus were all the same no matter using the primary or secondary blood sample. 1: The self-control well is negative. 2: Cells were all RhD-negative. 3: The IAT was negative after the cells were replaced with RhD-negative.
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