Abstract
Background
Rh antigens are critical in haemolytic disease of the foetus and newborn (HDFN). The D-- phenotype is a rare blood group characterised by the lack of expression of C, c, E and e antigens at the surface of red blood cells because of mutations in both RHCE alleles inactivating the expression of a “normal” protein. The aim of the study was to determine the molecular basis of D-- individuals of Indian origin.
Materials and methods
Ten Rh D-positive postnatal women who had produced antibodies against all Rh antigens, except D, leading to HDFN and foetal loss, were investigated. Extensive serological and molecular (polymerase chain reaction [PCR] using sequence-specific primers), quantitative multiplex PCR of short fluorescent fragments (QMPSF), and Sanger sequencing analyses were carried out.
Results
Serological testing with anti-C, anti-c, anti-E, and anti-e reagents showed absence of the four antigens in all ten index cases, as well as in three siblings. Flow cytometry indicated absence of these antigens with a typical exalted expression of the D antigen, thus confirming the rare D-- phenotype. Molecular analysis by QMPSF suggested homozygous CE-D hybrid alleles causing the D-- phenotype: RHCE-D(3–9)-CE (n = 11), RHCE-D(3–8)-CE (n=1), and RHCE-D(2–6)-CE (n=1).
Discussion
For the first time, we report the molecular basis of the D-- phenotype in the Indian population. Identification and characterisation of RHCE-null variants by molecular methods can help resolve transfusion-related problems in these individuals. Family studies of index cases helped to identify rare blood donors and offer counselling to females of child-bearing age on the complications involved in such pregnancies.
Keywords: rare blood group, RHD variants, D--, rearrangement, RHD-RHCE hybrid
INTRODUCTION
The Rh is the only known protein-based blood group system with more than 50 antigens1. The two RH genes, namely RHD and RHCE, hold >90% sequence similarity and are separated by 30 kilobases (kb) with an interspaced TMEM50A gene2,3. The RHD gene encodes only for D antigen. Conversely, the polymorphic RHCE gene encodes the antithetical C and c antigens on the one hand, and the antithetical E and e antigens on the other4. Alloantibodies produced against these five Rh antigens (D, C, c, E and e) are known to be involved in haemolytic disease of the foetus and newborn (HDFN) and haemolytic transfusion reaction (HTR)5–9.
Beyond the eight standard Rh haplotypes (i.e., DCe, Dce, dce, etc.) found at various frequencies in different populations10, rare Rh phenotypes, such as Rhnull and D--, encoded by unusual variant haplotypes, are rarely encountered in routine blood bank practice11. Rhnull phenotype is characterised by a complete absence of the D, C, c, E, and e antigens12. In the D-- phenotype, Rh antigens encoded by the RhCE protein, including C, c, E, and e, are all absent with a typical exalted D antigen expression to the extent that IgG anti-D reagents can agglutinate the red blood cells (RBCs) in the saline phase10.
In developing countries, such as in India, blood banks do not usually type for C, c, E and e antigens on a routine basis13. Thus D-- phenotype is commonly detected during antibody screening/identification or crossmatching in individuals who have developed alloantibodies against RhCE antigens or in a woman with a problematic obstetric history14,15. If sensitised with missing Rh antigens, these individuals form anti-Rh17 (or anti-Hro) alloantibody16. Therefore, given the scarcity of compatible donors, an individual with this rare phenotype poses a challenge to transfusion medicine. Conversely, blood units from donors with such rare phenotypes are the most valuable for transfusion purposes. Since the first report of D-- in 195117, several cases have been reported involving various mutational mechanisms in RHCE, including single nucleotide variations (SNVs), such as substitutions, insertions/deletions, and splice site mutations, and structural variants (hybrid genes)10. Here, we report for the first time the molecular bases of D-- phenotype in the Indian population.
MATERIALS AND METHODS
Patients and serological analysis
Ten RhD positive postnatal women with recurrent foetal loss were referred to the Department of Transfusion Medicine (ICMR-NIIH, Mumbai, India) for detailed laboratory evaluation from 2016 to 2019. Informed consent was obtained from all the subjects. ABO blood group was determined with both forward and reverse grouping. Samples were typed for D, C, c, E, and e antigens with antisera from four different manufacturers (Bio-Rad Laboratories, Irvine, CA, USA; Immucor Inc., Norcross, GA, USA; Tulip Diagnostics, Goa, India; Alba Bioscience Ltd., Penicuik, Scotland, UK). Absence of RhCE antigens was further confirmed by ether-elution technique. Direct antiglobulin test (DAT), and antibody screening and identification were performed according to the manufacturers’ instructions. The family members of the index cases available for extended investigation during the study period were also screened for the D-- phenotype.
Flow cytometric studies
Flow cytometric analysis for the expression of Rh antigens was carried out on a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA, USA) with the use of blood grouping monoclonal IgM anti-D, anti-C, anti-c, anti-E, anti-e reagents (BioRad, Diamed GmbH, Cressier, Switzerland) and in-house human polyclonal IgG anti-D. Anti-human globulin produced in goat (FITC-labelled polyvalent anti-human immunoglobulin [G, A, M], Sigma-Aldrich GmbH, Schnelldorf, Germany) was used as a secondary antibody. Appropriate control RBCs (R1R2, R1r, R2r, R1R1, R2R2, rr) were selected according to the Rh antigen being analysed. The test was carried out according to the procedure previously described18.
Molecular typing
DNA was extracted using the commercially available FlexiGene® DNA Extraction Kit (QIAGEN®, Hilden, Germany). Polymerase chain reaction using sequence-specific primers (SSP-PCR) was initially performed to confirm the absence of C, c, E and e antigens at the molecular level19. Copy number variations (CNVs) within the RHD and RHCE genes were further investigated by the quantitative multiplex PCR of short fluorescent fragments (QMPSF) according to the method described previously20. Briefly, all the exons were co-amplified by multiplex PCR in two separate tubes (i.e., one per gene) using FAM-labelled universal primers. Two control primer sets targeting the HFE and F9 genes were used along with exon-specific primers. The amplified products were run by capillary electrophoresis (Genetic Analyzer 3730xl, Applied Biosystems, Waltham, MA, USA) using the GeneScan 500 LIZ dye (Thermo Fisher Scientific, Waltham, MA, USA) as the size standard. Output data were analysed with GeneMapper software (Thermo Fisher Scientific). Presence of any sequence variations within the RHCE gene was assessed by the conventional Sanger sequencing method as previously described21.
RESULTS
Ten cases with a history of consecutive pregnancy loss and anaemia were referred to our institute for antibody identification. The first delivery of all except one woman was normal. However, during the second pregnancy, every foetus developed severe anaemia and jaundice. In one case, the first child died of HDN as the woman had been transfused with an ABO-compatible, Rh D-positive RBC unit in her childhood.
All referred samples displayed normal forward grouping, but reverse grouping showed the presence of an alloantibody. When tested with different clones of anti-D, -C, -c, -E and -e reagents, all samples only showed the presence of D antigen, suggesting that all CcEe antigens were absent. While performing RhD typing with IgG antisera, agglutination was observed in the saline phase depicting the exalted expression of D antigen. To confirm whether any Rh CcEe antigens were present in a weaker form, all samples along with known RBC controls (R1R1, R1r, R2r and rr) were adsorbed separately with commercial anti-C, -c, -E, and -e antibodies. After adsorption, ether-elution was performed to remove any bound antibody. In all cases, adsorption/elution test on patients’ RBCs confirmed the total absence of Rh CcEe antigens, while controls showed positive reaction (data not shown). Antibody study showed panagglutination reaction, suggesting the presence of alloantibodies. In all cases, DAT was negative. The antibody titre in D-- cases ranged from 1: 4 to 1: 64 in IgM phase and 1: 64 to 1: 512 in IgG phase. Family history revealed that, in four families at least, the parents had consanguineous marriages. Out of 41 family members of seven probands, three siblings showed absence of Rh CcEe antigens. Detailed serological studies indicated that all ten women and their three siblings belong to the very rare D-- blood group.
Flow cytometry analysis also showed absence of the CcEe antigens on proband RBCs, while control RBCs with ryr/r′r″ phenotype were positive (Figure 1). In addition, exalted expression of D antigen in a homozygous D-- individual was observed when compared with R1R1 or heterozygous D-- controls (R2/D-- and R1/D--). Overall, flow cytometry analysis confirmed the D-- phenotype.
Figure 1.
Flow cytometric analysis
(A) Positive control (ryr/r′r″) for C, c, E, and e antigen expression. (B) Individual with a D-- phenotype showing the absence of the C, c, E, and e antigen expression. (C) Homozygous D-- individual showing exalted D antigen expression when compared with R1R1 individual and heterozygous D-- individuals.
While molecular screening by SSP-PCR specific for polymorphic sites encoding C/c and E/e antigens was negative, the presence of CNVs in RHD and RHCE potentially defining hybrid genes was investigated by QMPSF as described previously. In all index cases (n=10), gene conversion events between RHCE and RHD were shown, defining the RHCE-D(3–9)-CE (n=8), RHCE-D(3–8)-CE (n=1), and RHCE-D(2–6)-CE (n=1) hybrid genes resulting in the rare D-- phenotype when present at the homozygous state (Figure 2). When a total of 41 family members of the seven probands were screened, three more individuals with the D-- phenotype were identified; all presented with the RHCE-D(3–9)-CE rearrangement at the homozygous state in accordance with the variant allele identified in their respective families (Table I).
Figure 2.
RHCE QPMSF analysis in Indians with a D—phenotype
*Undetected RHCE exons.
Table I.
Summary of serology, molecular and family studies of index D-- cases
| Case n. | ABO blood group and Rh phenotype | Molecular background | N. of family members screened | Family member with D--/D--phenotype |
|---|---|---|---|---|
| 1 | B D--/D-- | RHCE-D(3–8)-CE | 0 | 0 |
| 2 | A D--/D-- | RHCE-D(2–6)-CE | 5 | 0 |
| 3 | O D--/D-- | RHCE-D(3–9)-CE | 9 | 0 |
| 4 | B D--/D-- | RHCE-D(3–9)-CE | 8 | 1 (AB D--/D--) |
| 5 | B D--/D-- | RHCE-D(3–9)-CE | 4 | 0 |
| 6 | A D--/D-- | RHCE-D(3–9)-CE | 3 | 1 (B D--/D--) |
| 7 | B D--/D-- | RHCE-D(3–9)-CE | 9 | 1 (B D--/D--) |
| 8 | A D--/D-- | RHCE-D(3–9)-CE | 0 | 0 |
| 9 | B D--/D-- | RHCE-D(3–9)-CE | 0 | 0 |
| 10 | A D--/D-- | RHCE-D(3–9)-CE | 3 | 0 |
| Total | 41 | 3 |
A detailed family study of two probands was further performed. CNV analysis of their parents revealed the presence of the RHD gene at the homozygous state with a single functional copy of the RHCE gene. As in the parents, some of the siblings were found to carry the D-- haplotype at the heterozygous state (Figure 3).
Figure 3.
Pedigree analysis of two families with two D-- cases showing the presence of heterozygous D-- allele in parents
Arrows: index cases.
DISCUSSION
The proximity and tail-to-tail orientation of the highly homologous RHD and RHCE genes promote gene rearrangement during crossover3. Beside numerous SNVs in both genes, the genetic mechanisms involved in the expression of Rh variants include deletion or rearrangement of some or entire coding regions between these two genes. Some of the Rh-deficient phenotypes, including Rhnull and D--, are the outcome of this phenomenon. Most potent forms of Rh deficiency, which may cause RBC disorders (stomatocytosis, spherocytosis, etc.), are Rhnull (complete absence of the RhD and RhCE proteins at the RBC surface) and Rhmod (severe decrease in the expression of both proteins)22. Other haplotypes causing Rh deficiency lacking E/e antigens include Dc-, DCw-, D--, D··, and DIV(C)-10.
The D-- phenotype is characterised by the lack of expression of C, c, E and e antigens at the surface of the RBCs. This phenotype is strongly linked to consanguinity and has been found in diverse populations with an estimated frequency of 0.0005, 0.0047, and 0.0032, in Sweden, Iceland, and Japan, respectively10. While incidence of the D-- phenotype in Indians has not been studied so far, not a single case of Rh-deficient phenotype has been discovered in our institute (i.e., ICMR-NIIH, Mumbai, India) after typing more than 30,000 individuals for Rh antigens, which emphasises the rarity of these blood groups in India. Ethnic diversity was observed in individuals with D-- phenotype. Five D-- cases belonged to Muslims and the remaining five individuals were from the Hindu community. Distribution according to geographical region showed that the majority of the cases (n=5) were reported from the western part of India, followed by north (n=2), south (n=2) and east (n=1) part of the country. Although the molecular basis of D-- phenotype expression remained unidentified in a few reports23,24, different mutational mechanisms that alter the RHCE locus resulting in this rare phenotype have been reported (Table II). These mutations include: non-functional hybrid genes (as identified here)24–30, large deletions31,32, and SNVs30,33. As hybrid genes have been commonly reported, QMPSF analysis was carried out preferentially for the molecular characterisation of D-- individuals.
Table II.
Molecular mechanisms of individuals with D-- phenotype in this study and in various populations
| RHCE allele | Origin/ethnicity | Reference |
|---|---|---|
| RHCE-D(3–9)-CE | Indian | This study |
| RHCE-D(3–8)-CE | Indian | This study |
| RHCE-D(2–6)-CE | Indian | This study |
| Apparent wild-type RHCE | Caucasian (France) | Chérif-Zahar B et al., 23 |
| Apparent wild-type RHCE | Native Americans (Iroquois) | Kemp TJ et al., 24 |
| RHCE-D(3–9)-CE | Caucasian (Italy, Iceland, UK) | Kemp TJ et al.,24 |
| RHCE-D(3–8)-CE | Caucasian (Italy, UK) | Kemp TJ et al.,24 |
| RHD(1–9)-CE | Caucasian (Italy) | Chérif-Zahar B et al.,25 |
| RHCE-D(3–7)-CE | Caucasian | Cheng GJ et al.,26 |
| RHCE-D(3–7)-CE | East-Asian (Japan) | Okuda H et al.,27 |
| RHCE-D(3–8)-CE | Asia (Malaysia) | Flatt JF et al.,29 |
| RHCE-D(4–9)-CE | Asia (Malaysia) | Flatt JF et al.,29 |
| RHCE*Ce(c.87insT) | Caucasian | Ochoa-Garay G et al.,30 |
| RHCE*Ce(c.148+5G>A) | Caucasian (Turkey) | Ochoa-Garay G et al.,30 |
| RHCE*cE(c.221G>A) | Caucasian (Cajun) | Ochoa-Garay G et al.,30 |
| RHCE-D(3–8)-CE | East-Asian (South-Korea) | Ochoa-Garay G et al.,30 |
| RHCE-D(3–9)-CE | Hispanic | Ochoa-Garay G et al.,30 |
| RHCE*cE(c.907delC) | Hispanic | Westhoff CM et al.,33 |
Three large rearrangements that have already been reported previously were observed: RHCE-D(3–9)-CE, RHCE-D(3–8)-CE, and RHCE-D(2–6)-CE. In our study, the RHCE-D(3–9)-CE hybrid allele, also found in Caucasians24,30, was the most common(n=11) cause of the D-- phenotype.
The RHCE-D(3–8)-CE hybrid allele has been reported in Europe and in individuals of Malaysian D-- origin24,29. Finally, we observed the RHCE-D(2–6)-CE rearrangement in one index D-- case. A similar rearrangement had previously been observed in an individual with the D·· (Evans) phenotype26. Additional testing will be carried out to conclusively confirm the phenotype in this latter individual.
Interestingly, family screening of index cases helped to identify three more D-- members. These members can be registered as future blood donors as they have not developed any alloantibody. Unfortunately, family members of three D-- index cases could not be contacted.
CONCLUSIONS
Molecular mechanisms involving gene rearrangement between RHD and RHCE is responsible for D-- phenotype in Indians. We found QMPSF to be an invaluable tool in detecting Rh variants formed by hybridisation. Family studies should be performed to detect similar variants in other family members who could be blood donors for an individual with this rare phenotype. Identification of rare phenotypes and their characterisation by molecular methods can help resolve transfusion-related problems in such individuals. This warrants the need to develop a national rare donor registry for better transfusion management of transfusion-dependent patients.
Footnotes
FUNDING AND RESOURCES
This work was supported by the Indo-French Centre for the Promotion of Advanced Research/Centre Franco-Indien pour la Promotion de la Recherche Avancée (IFCPAR/CEFIPRA, Project 5203-1), the ICMR-National Institute of Immunohaematology (ICMR-NIIH), the Etablissement Français du Sang (EFS)-Bretagne, and the Institut National de la Santé et de la Recherche Médicale (Inserm).
AUTHORSHIP CONTRIBUTIONS
SK and YF designed the research study. SK, DG, AKB, SPS, AC, and RS contributed to sample management. GM, HG, DP and YF carried out the experiments. SK,GM and YF wrote the manuscript. MM and CF revised and approved the manuscript.
The Authors declare no conflicts of interest.
REFERENCES
- 1.Reid ME, Lomas-Francis C, Olsson ML Rh Blood Group System. The Blood Group Antigen FactsBook. 3rd ed. London, UK: Elsevier Ltd; 2012. pp. 147–262. [Google Scholar]
- 2.Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet. 2001;2:10. doi: 10.1186/1471-2156-2-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apheres Sci. 2011;44:81–91. doi: 10.1016/j.transci.2010.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Mouro I, Colin Y, Chérif-Zahar B, et al. Molecular genetic basis of the human Rhesus blood group system. Nat Genet. 1993;5:62–65. doi: 10.1038/ng0993-62. [DOI] [PubMed] [Google Scholar]
- 5.Urbaniak SJ, Greiss MA. RhD haemolytic disease of the fetus and the newborn. Blood Rev. 2000;14:44–61. doi: 10.1054/blre.1999.0123. [DOI] [PubMed] [Google Scholar]
- 6.Hackney DN, Knudtson EJ, Rossi KQ, et al. Management of pregnancies complicated by anti-c isoimmunization. Obstetr Gynecol. 2004;103:24–30. doi: 10.1097/01.AOG.0000109206.22354.2C. [DOI] [PubMed] [Google Scholar]
- 7.Bowman JM, Pollock JM, Manning FA, Harman CR. Severe anti-C hemolytic disease of the newborn. Am J Obstetr Gynecol. 1992;166:1239–43. doi: 10.1016/s0002-9378(11)90614-0. [DOI] [PubMed] [Google Scholar]
- 8.Joy SD, Rossi KQ, Krugh D, O’Shaughnessy RW. Management of pregnancies complicated by anti-E alloimmunization. Obstetr Gynecol. 2005;105:24–28. doi: 10.1097/01.AOG.0000149153.93417.66. [DOI] [PubMed] [Google Scholar]
- 9.Chapman J, Waters AH. Haemolytic disease of the newborn due to Rhesus anti-e antibody. Vox Sang. 1981;41:45–47. doi: 10.1111/j.1423-0410.1981.tb01011.x. [DOI] [PubMed] [Google Scholar]
- 10.Daniels G. Human Blood Groups. 3rd ed. Oxford, UK: Blackwell Publishing Ltd; 2013. Rh and RHAG blood group systems; pp. 182–258. [Google Scholar]
- 11.Howard PR. Basic & Applied Concepts of Blood Banking and Transfusion Practices. 4th ed. Elsevier Health Sciences; 2016. [Google Scholar]
- 12.Levine P, Celano MJ, Vos GH, Morrison J. The first human blood, ---/---, which lacks the ‘D-like’ antigen. Nature. 1962;194:304–05. doi: 10.1038/194304a0. [DOI] [PubMed] [Google Scholar]
- 13.Lamba DS, Kaur R, Basu S. Clinically significant minor blood group antigens amongst North Indian donor population. Adv Hematol. 2013 doi: 10.1155/2013/215454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Denomme GA, Ryan G, Seaward PGR, et al. Maternal ABO-mismatched blood for intrauterine transfusion of severe hemolytic disease of the newborn due to anti-Rh17. Transfusion. 2004;44:1357–60. doi: 10.1111/j.1537-2995.2004.04082.x. [DOI] [PubMed] [Google Scholar]
- 15.Abolhassan Choobdar F, Milani H, Behrouzi K, et al. Anti-Rh17 alloimmunization: a rare case of severe hemolytic disease of the newborn and review of the literature. J Pediatr Rev. 2020;8:29–34. [Google Scholar]
- 16.Chowdhry M, Agrawal S, Thakur UK. A case of rare anti-Hro alloantibody in a tertiary care center in India. Asian J Transfus Sci. 2019;13:54–56. doi: 10.4103/ajts.AJTS_26_18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Race RR, Sanger R, Selwyn JG. A possible deletion in a human Rh chromosome: a serological and genetical study. Br J Exp Pathol. 1951;32:124–35. [PMC free article] [PubMed] [Google Scholar]
- 18.Kulkarni S, Mohanty D, Gupte S, et al. Flow cytometric quantification of antigen D sites on red blood cells of partial D and weak D variants in India. Transfus Med. 2006;16:285–89. doi: 10.1111/j.1365-3148.2006.00667.x. [DOI] [PubMed] [Google Scholar]
- 19.Kulkarni S, Choudhary B, Gogri H, et al. Molecular genotyping of clinically important blood group antigens in patients with thalassaemia. Ind J Med Res. 2018;148:713–20. doi: 10.4103/ijmr.IJMR_455_17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Fichou Y, Le Maréchal C, Bryckaert L, et al. A convenient qualitative and quantitative method to investigate RHD-RHCE hybrid genes. Transfusion. 2013;53:2974–82. doi: 10.1111/trf.12179. [DOI] [PubMed] [Google Scholar]
- 21.Fichou Y, Le Maréchal C, Férec C. The RHD*weak D type 4.0 allele is predominantly but not exclusively cis-associated with the altered RHCE*ce(c.48C, c.105T, c.733G, c.744C, c.1025T) allele in the French population. Transfus Med. 2014;24:120–22. doi: 10.1111/tme.12100. [DOI] [PubMed] [Google Scholar]
- 22.Cartron JP. Rh-deficiency syndrome. Lancet. 2001;358:S57. doi: 10.1016/S0140-6736(01)07069-6. [DOI] [PubMed] [Google Scholar]
- 23.Chérif-Zahar B, Raynal V, D’Ambrosio AM, et al. Molecular analysis of the structure and expression of the RH locus in individuals with D--, Dc-, and DCw- gene complexes. Blood. 1994;84:4354–60. [PubMed] [Google Scholar]
- 24.Kemp TJ, Poulter M, Carritt B. A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D--phenotype. Am J Hum Genet. 1996;59:1066–73. [PMC free article] [PubMed] [Google Scholar]
- 25.Cherif-Zahar B, Raynal V, Cartron JP. Lack of RHCE-encoded proteins in the D-- phenotype may result from homologous recombination between the two RH genes. Blood. 1996;88:1518–20. [PubMed] [Google Scholar]
- 26.Cheng GJ, Chen Y, Reid ME, Huang CH. Evans antigen: a new hybrid structure occurring on background of D?? and D-- Rh complexes. Vox Sang. 2000;78:44–51. doi: 10.1159/000031148. [DOI] [PubMed] [Google Scholar]
- 27.Okuda H, Fujiwara H, Omi T, et al. A Japanese propositus with D-- phenotype characterized by the deletion of both the RHCE gene and D1S80 locus situated in chromosome 1p and the existence of a new CE-DCE hybrid gene. J Hum Genet. 2000;45:142–53. doi: 10.1007/s100380050201. [DOI] [PubMed] [Google Scholar]
- 28.Huang CH, Peng J, Chen HC, et al. RH locus contraction in a novel Dc-/D-- genotype resulting from separate genetic recombination events. Transfusion. 2004;44:853–59. doi: 10.1111/j.1537-2995.2004.03324.x. [DOI] [PubMed] [Google Scholar]
- 29.Flatt JF, Musa RH, Ayob Y, et al. Study of the D-- phenotype reveals erythrocyte membrane alterations in the absence of RHCE. Br J Haematol. 2012;158:262–73. doi: 10.1111/j.1365-2141.2012.09149.x. [DOI] [PubMed] [Google Scholar]
- 30.Ochoa-Garay G, Moulds JM, Cote J, et al. New RHCE variant alleles encoding the D-- phenotype. Transfusion. 2013;53:3018–23. doi: 10.1111/trf.12404. [DOI] [PubMed] [Google Scholar]
- 31.Blunt T, Steers F, Daniels G, Carritt B. Lack of RH C/E expression in the Rhesus D-- phenotype is the result of a gene deletion. Ann Hum Genet. 1994;58:19–24. doi: 10.1111/j.1469-1809.1994.tb00722.x. [DOI] [PubMed] [Google Scholar]
- 32.Huang CH, Reid ME, Chen Y. Identification of a partial internal deletion in the RH locus causing the human erythrocyte D-- phenotype. Blood. 1995;86:784–90. [PubMed] [Google Scholar]
- 33.Westhoff CM, Vege S, Nickle P, et al. Nucleotide deletion in RHCE*cE(907delC) is responsible for a D-- haplotype in Hispanics. Transfusion. 2011;51:2142–47. doi: 10.1111/j.1537-2995.2011.03144.x. [DOI] [PubMed] [Google Scholar]