Review. Clinical and Laboratory Update on the DEL Variant. Rh Blood Group System

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1 Clinical and Laboratory Update on the DEL Variant Pornlada Nuchnoi, PhD, 1,2* Jairak Thongbus, MSc, 1,3 Apapan Srisarin, MSc, 1 Usanee Kerdpin, PhD, 4 Virapong Prachayasittikul, PhD 5 Lab Med Fall 2014;45: DOI: /LMTUZ00O7VFTGCEB ABSTRACT Serological assays for the RhD blood group are based on detection of the RhD antigen on human red blood cells using a specific anti-d antibody. The weak expression of the RhD antigen in the DEL variant hinders the sensitivity of conventional serological assays. Evidence of anti-d immunization in patients with D-negativity who have received DEL-variant blood units has been reported in various populations. This observation has prompted the need for genetic epidemiological and Since the discovery of the Rh blood group system in 1940, RhD has been considered the most highly polymorphic blood group. 1 In addition to the RhD-positive (Rh+) and RhD-negative (Rh-) phenotypes, qualitative (partial D) and quantitative (weak D and DEL) variants have become increasingly important in the practice of transfusion medicine. The weak expression of RhD antigen, termed DEL or D elute (D el ), was first reported in DEL is the RhD blood group variant most commonly reported in the Asian population, particularly in individuals of Japanese and Chinese ethnicity. The prevalence of DEL variant is approximately 30% in Asian RhD-negative donors and 0.1% in white RhDnegative donors. The DEL variant is phenotypically similar to the RhD-negative phenotype and is routinely identified Abbreviations D el, D elute; RBC, red blood cell; AHG, antihuman globulin; SNPs, single-nucleotide polymorphisms; IAT, indirect-antihuman globulin test; HTR, hemolytic transfusion reaction; PCR-SSP, polymerase chain reaction sequence-specific primer; DHTR, delayed hemolytic transfusion reaction; HDFN, hemolytic disease of the fetus and newborn; MoAbs, monoclonal antibodies 1 Department of Clinical Microscopy; 2 Center for Innovation Development and Technology Transfer, Mahidol University, Bangkok, Thailand; 3 National Blood Center, Thai Red Cross Society, Bangkok, Thailand, 4 Department of Chemistry, Naresuan University, Phitsanulok, Thailand; 5 Department of Clinical Microbiology and Applied Technology, Mahidol University, Bangkok, Thailand *To whom correspondence should be addressed. pornlada.nuc@mahidol.ac.th clinical data on the DEL variant in the development of DEL molecular diagnostic testing. This review highlights the molecular features of the DEL variant, the clinical consequences of DEL-blood transfusion, and current approaches for detection of the DEL-variant for donor screening and transfusion. Keywords: RHD, DEL, genetic variation, blood group genotyping, transfusion reaction, transfusion medicine as RhD negative by conventional serological tests. The number of D-antigen sites per DEL red blood cell (RBC) is estimated to be less than 22 compared with the 30,000 antigen sites for normal D and 1500 to 7000 sites for weak D. 3-5 Consequently, the DEL variant is nonreactive in the antihuman globulin (AHG) test; this is different from the conventional weak D variant, which produces a positive screening result. Therefore, DEL variant is only detected by the adsorption-elution test (Figure 1). 6 The majority of current DEL research has focused on identifying the associations between the identification of DEL-associated alleles and alloimmunization in various populations. This review provides an update of the clinical considerations associated with the DEL variant. Rh Blood Group System The RHD (OMIM#111680) and RHCE (OMIM#111700) genes encode the D and CE proteins/antigens in the Rh blood group system. The RH locus spans approximately 60 kb of each gene in the opposite orientation on chromosome 1p36.11 (Figure 2). RHD and RHCE are highly homologous, with 93.8% identity between the entire exon and intron regions of the two genes. Each RH gene is composed of 10 exons that encode 417 amino acid proteins. The RhD and RhCE proteins differ by 32 to 35 amino acids, depending on the RHCE allele. In the RhD-negative phenotype the RHD region is deleted. However, approximately 67% of RhD-negative ethnic Africans harbor at Fall 2014 Volume 45, Number 4 Lab Medicine 285

2 DEL -Negative for indirect anti-globulin test -Positive for absorbtion-elution test -Presence of RHD gene (no RHD deletion) -DEL associated alleles in specific ethnic groups such as K409K (1227G >A) in Asian least 1 copy of RHD*ψ, which is an inactivated RHD that results from a 37-bp duplication in exon 4 and a nonsense mutation in exon 6. 7 It has been noted 8-9 that DEL variant is usually linked to RhCE*Ce or RhCE*cE. The complexity and heterogeneity of these genetic variations supports the idea that RHD is a highly polymorphic gene that causes RhD variants. Molecular Alleles of DEL Variant in Different Ethnicities RhD variants result from single-nucleotide polymorphisms (SNPs) 10 and genetic recombination, resulting in an RHD- CE-D hybrid gene SNPs found in RHD cause protein structural changes (partial-d) and heterogeneity in expression level (weak D and DEL). However, the molecular mechanisms underlying the DEL variant remain unclear. RHD 1227A (K409K) is the most common SNP found in ethnic Eastern Asians with DEL. 14 K409K is associated with a splice site in exon 9 of RHD. 10 The mechanism underlying the DEL variant is mainly explained through nucleotide alterations in the transmembrane and cytosolic domains of the RhD protein. A large number of DEL-associated alleles have been identified across ethnic populations. In whites, the most common DEL alleles include Met295Ile and IVS3+1 g>a (splice site mutation). 3,6,11 Several RHD SNPs (ie, DEL-associated alleles) have been reported with different frequencies among various populations (Table 1). The complexity of RHD challenges the detection of RHD architecture and modifier genes in DEL with different genetic backgrounds Clinical Consequences of DEL Variant in Transfusion Practice The immunogenicity of the D antigen on DEL RBCs is uncertain There are evidences 3,18-19 that DEL alleles exert different characteristics. The number of D antigen sites on RBCs varies among DEL alleles. Although the number Figure 1 Characteristics of the DEL variant of antigen sites on DEL RBCs is extremely low, anti-d production in patients with D-negativity who received a DEL-positive transfusion has been observed in various populations. The evidence 20 shows that DEL blood transfusion increases the risk of secondary immunization in individuals with existing anti-d. We note the evidence of potential risk of primary immunization by DEL blood transfusion per in the following reported cases. 1. The first reported case of allo-anti-d production occurred in a 58-year-old white woman (dce/dce genotype) who received a transfusion containing DEL RBCs (Ce/ce). The patient had no history of transfusion or transplantation; she had had 1 pregnancy, resulting in the live birth of an RhD-negative child. Anti-D antibodies were detected 3 days after transfusion. An adsorption-elution test was used to confirm that the donor harbored the DEL variant. The molecular screening tests revealed that the patient had a normal RHD allele. The RHD 1227A allele (K409K) was excluded. Sequencing analysis at the promoter, coding, and intron regions revealed the deletion of 4 nucleotides (CTCT) in intron 5 (RHD IVS5-38del4). One year later, the anti-d titer in the patient was 1:128 and 1:64 via the gel card and test tube techniques, respectively. The blood specimen from this donor was RhD negative. An additional 2 patients with RhD negativity received 3 units each of packed DEL RBCs. A specimen from 1 patient tested positive for indirect-antihuman globulin test (IAT) 4 weeks after transfusion without specific antibody identification An increased anti-d titer following transfusion of DEL positive units was reported in a 67-year-old Japanese woman. The woman was RhD-negative; primary immunization resulted from a transfusion of RhD-positive blood. However, the anti-d disappeared before From 2000 to 2003, the patient received crossmatch compatible RBC from 59 donors with RhD-negative for treatment of persistent intestinal bleeding. Among the specimens from 59 donors with RhD negativity, 2 were molecularly genotyped as DEL (K409K; 1227 G>A). The anti-d titer had increased from 1:8 in 2001 to 1:128 in There was no history of an acute or delayed 286 Lab Medicine Fall 2014 Volume 45, Number 4

3 Upstream rhesus box Downstream rhesus box SMP Figure 2 Architecture of RHD and RHCE loci. RHD hemolytic transfusion reaction (HTR). The results of this study suggested that the transfusion of DEL RBCs could trigger and boost the secondary immune response in recipients with RhD negativity who have a history of anti-d production The first case of primary allo anti-d immunization due to a DEL (RHD K409K) transfusion was reported in a 68-year-old ethnic Korean man. He received a transfusion of 1 unit of DEL RBCs; there was no evidence of acute or delayed HTR. The authors who reported the case detected anti-d in this patient 9 days after transfusion In 2009, Shao et al 23 reported transfusion of DEL-positive packed RBCs in 11 of 30 ethnic Chinese recipients of RhD-negative blood. All of the transfused DEL blood was typed as ethnic Asian (1227 G>A). Among the 11 recipients with RhD negativity, 2 were typed as heterozygous for DEL (DEL/dCcee) by polymerase chain reaction sequence specific primer (PCR-SSP). In 3 subject individuals with apparent anti-d positivity before transfusion (2 from a child with RhD positivity and 1 from a transfusion of RhD-positive blood), 2 of the subjects developed a higher anti-d titer after being transfused with 2 units of DEL blood (anti-d titer 1:8 to 1:64), and 1 subject continued to have a higher anti-d titer (1:512). The researchers also observed delayed hemolytic transfusion reaction (DHTR) in these 2 cases (titers: 1:64 and 1:512). Seven patients who had previously tested anti-d negative before transfusion (2 with the DEL variant) now tested anti-d positive, with a titer of 1:2 in 1 recipient. As we expected, the researchers detected no anti-d production in the 2 recipients with a DEL variant. This study highlights the potential effects of DEL transfusion induced anti-d in primary and secondary alloimmunization. Also, fetuses with DEL variant have a high potential for inducing anti-d production in mothers with true D-negativity. 23 RHCE Most individuals carrying DEL-associated alleles show no evidence of anti-d production. However, anti-d production has been demonstrated in an ethnic Austrian with an RHD (IVS3+1G>A) allele. 3X The report noted mild hemolytic disease of the fetus and newborn (HDFN) caused by IVS3+1G>A. Epitope mapping using different clones of anti-d monoclonal antibodies (MoAbs) revealed a lack of some D epitopes (partial D antigen). Therefore, this allele is associated with a partial DEL phenotype. 3,24 Laboratory Investigation of DEL Variant Standard serological tests for RhD commonly misidentify patients with DEL as having RhD negativity. The DEL variant exhibits extremely weak D antigen expression. Therefore, the genotyping assay is the current method of choice for DEL-variant detection. 25 DEL-associated alleles have been reported in various populations. 26,27 Therefore, the spectrum of DEL genotypes in each population should be considered to optimize a DEL genotyping strategy in these ethnic groups. DEL is highly associated with the C + phenotype (CDe or cde haplotypes) (Table 2). The RhCE phenotype distribution is comparable among ethnic Asians. 6,10,17,28,29 The Ccee phenotype is the major RHCE phenotype found in Asian (approximately 83% of population). Hence, the integration of RHD K409K genotyping and C antigen typing is a useful approach for DEL variant screening of RhD-negative Asian donors. 30,31 Genotyping of DEL variants is based on PCR Laboratories commonly use PCR-SSP followed by sequencing to detect known DEL alleles and for DEL allele screening. Recently, a molecular assay for DEL variant screening in serologically negative RhD patients has been suggested in the blood donor (Figure 3). 6,32 This molecular assay has Fall 2014 Volume 45, Number 4 Lab Medicine 287

4 Table 1. Distribution of DEL-Associated Alleles Among Different Ethnic Groups DEL-Carrying Individuals Per Ethnic Group, No. DEL Allele Taiwanese (n = 94) 10 Chinese (n = 26) 9 Chinese (n = 279) 17 Danish (n = 13) German (n=47) 6 Normal RhD 1 K409K IVS7+152A 1 a Hybrid allele b 3 1 3G>A 4 R10W 1 L18P 1 L84P 1 A137E 1 L153P 1 G212R 1 Y401X 1 X418L 2 IVS2-2A>G 2 IVS8+1G>A 1 659delG 2 885G>T T>C 1 IVS7-2A>C 1 IVS3+1G>A _94insT delA 4, not applicable. a Heterozygote of K409K and IVS7+152A. b RHD-CE-D hybrid. been widely used in Central Europe as well. Four PCR-SSP methods specific for intron 4, exon 7, exon 10 and promoter protocols have been effective for distinguishing the true D- negative genotype from the DEL variant. The RHD-CE-D hybrid allele may not be detected by PCR-SSPs specific for intron 4 and exon 7. RhD antigens were no expressed in the absence of these alleles. Additional molecular markers for DEL variant genotyping should be adjusted to the specific population studied, although the adsorption-elution test is an additional serological tool that we recommend for detection of DEL-variants. However, identification of DEL-variants may be hampered by technical errors, limitations in the anti-d MoAb reagents, and differences in protocols among laboratories. PCR for RHD PCR/genotyping is practical and useful for identifying DEL-variants. 32,36,37 Moreover, researchers 38 have applied the plasma cross pooling strategy for RHD gene detection. RHD gene fragments in intron 4, exon 7, and exon 10 have been used as RHD-specific markers for RHD carriers. It has been shown 38 that the plasma cross pooling strategy is a cost effective and reliable approach identifying individuals carrying RhD-negative RHD genes. Plasma DNA has also been used to identify individuals with RHD positivity, and this strategy might be an alternative approach for large blood-service and obstetrics centers. 8 Table 2. RHCE Phenotypic Distribution of DEL Among Distinct Ethnic Populations Population Frequency RHCE Phenotype Chinese 17 Taiwanese 10 Taiwanese 28 German 6 Ccee 83.51% 83% 83.3% 82.97% CCee 12.19% 15% 14.3% 10.65% CCEe 0.36% 1% 0.8% CcEe 3.23% 1% 1.6% ccee 0.72% 6.38% ccee 2.13%..., not applicable. Researchers have developed a DEL-variant molecular-screening strategy to be used in donors with RhD negativity, particularly females of childbearing age. In Central Europe, transfusion services routinely screen for DEL-variants. 32 This procedure should be adopted in Asia as part of the first-time donor-testing policies of blood banks, hospitals, and other relevant health care facilities to ensure the transfusion of safe blood to patients. 41 DEL variant blood can be safely transfused to recipients who test RhD positive. A retrospective study of DEL-transfused 288 Lab Medicine Fall 2014 Volume 45, Number 4

5 Figure 3 PCR-SSP for promoter and exon 10 negative positive RHD deletion blood units is clinically important for investigating the potential for immunization of recipients who test RhD negative. Avoiding transfusion of blood from DEL-variant donors into patients with true RhD negativity should lower the incidence of HTR. 42,43 Conclusion Serological D-negative PCR-SSP for intron 4 and exon 7 negative positive RHD hybrid K409K RHD K409K non-k409k D-negative DEL Weak-D Schematic of molecular approach for RHD genotyping for distinguishing D-negative, DEL, and weak D in the ethnic Asian population. Anti-D immunization in recipients who are apparently RhD negative is a concern in management of donors who carry the DEL variant. The heterogeneity of DEL genotypes results in highly distinct and ethnically-specific molecular expression of this antigen. Elucidation of the molecular genetics underlying DEL variant is clinically beneficial for the development of DEL-variant screening guidelines in specific populations. Comprehensive profiling of common DEL-associated alleles is needed to prevent avoidable immunization of susceptible patients. The combination of C + phenotyping and molecular assays for DEL variants are cost-effective, reliable approaches for first-time and repeat donors in blood-service centers. The establishment of a policy for DEL variant screening will be clinically useful for transfusion practices. Acknowledgments This work was supported by the Thailand Research Fund, Office of the Higher Education Commission and Mahidol University to PN (MRG ). This work was also partially supported by Office of the Higher Education Commission and Mahidol University under the National Research University Initiatives. LM References 1. Wagner FF, Kasulke D, Kerowgan M, Flegel WA. Frequencies of the blood groups ABO, Rhesus, D category VI, Kell, and of clinically relevant high-frequency antigens in south-western Germany. Infusionsther Transfusionsmed. 1995;22(5): Okubo Y, Yamaguchi H, Tomita T, Nagao N. A D variant, D el? Transfusion. 1984;24(6):542. DOI: /j x. 3. Körmöczi GF, Gassner C, Shao CP, Uchikawa M, Legler TJ. A comprehensive analysis of DEL types: partial DEL individuals are prone to anti D alloimmunization. Transfusion. 2005;45(10): Kulkarni S, Mohanty D, Gupte S, Vasantha K, Joshi S. Flow cytometric quantification of antigen D sites on red blood cells of partial D and weak D variants in India. Transfus Med. 2006;16(4): Gorick B, McDougall DC, Ouwehand WH, et al. Quantitation of D sites on selected weak D and partial D red cells. Vox Sang. 1993;65(2): Flegel 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(3): Singleton, BK, Green CA, Avent ND, 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(1): Flegel WA, Wagner FF. Molecular biology of partial D and weak D: implications for blood bank practice. Clin Lab. 2002;48(1-2): Shao C-P, Maas J-H, Su Y-Q, Köhler M, Legler TJ. Molecular background of Rh D-positive, D-negative, D el and weak D phenotypes in Chinese. Vox Sang. 2002;83(2): Chen J-C, Lin T-M, Chen YL, Wang Y-H, Jin Y-T, Yue C-T. RHD 1227A is an important genetic marker for RhD el individuals. Am J Clin Pathol. 2004;122(2): Luettringhaus TA, Cho D, Ryang DW, Flegel WA. An easy RHD genotyping strategy for D- East Asian persons applied to Korean blood donors. Transfusion. 2006;46(12): Okuda H, Kawano M, Iwamoto S, et al. The RHD gene is highly detectable in RhD-negative Japanese donors. J Clin Invest. 1997;100(2): Sun C-F, Chou C-S, Lai N-C, Wang W-T. RHD gene polymorphisms among RhD-negative Chinese in Taiwan. Vox Sang. 1998;75(1): Chen Q, Li M, Li M, et al. Molecular basis of weak D and DEL in Han population in Anhui Province, China. Chin Med J (Engl). 2012;125: Peng C-T, Shih M-C, Liu T-C, Lin I-L, Jaung S-J, Chang J-G. Molecular basis for the RhD negative phenotype in Chinese. Int J Mol Med. 2003;11(4): Cherif-Zahar B, Raynal V, Gane P, et al. Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency. Nat Genet. 1996;12(2): Fall 2014 Volume 45, Number 4 Lab Medicine 289

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