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Genetic basis of rare blood group variants

Wigman, L.

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Citation for published version (APA):Wigman, L. (2013). Genetic basis of rare blood group variants.

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Download date: 03 Jun 2020

Chapter 4

Characterization of known and novel RHD variant alleles in 37,764 Dutch D- pregnant women

Lonneke Haer-Wigman1*

Tamara C. Stegmann1*

Florentine F. Thurik1

Renate Bijman1

Bernadette Bossers2

Goedele Cheroutre2

Remco Jonkers2

Peter Ligthart2

Barbera Veldhuisen1,2

Masja de Haas1,2

C. Ellen van der Schoot1

1 Sanquin Research, Amsterdam and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

2 Sanquin Diagnostic Services, Amsterdam, The Netherlands

Manuscript in preparation

Chapter 4

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Abstract

Background: In the Netherlands D- pregnant women are offered a quantitative fetal RHD

genotyping assay to determine the RHD status of their fetus to guide anti-D prophylaxis. We characterized maternal RHD variant alleles recognized in this genotyping assay. Methods: In 1.3% of the 37,764 D- pregnant women who were tested with the quantitative fetal RHD genotyping assay, targeting exon 5 and 7, a maternal variant allele was suspected and serological and molecular analysis was performed.

Results: In 0.96% (95% CI 0.86% - 1.05%) of the Dutch D- pregnant women a variant allele is present and 47% of these women carry the RHD*Ψ allele. Forty-five different RHD variant alleles were detected, including fourteen novel alleles. Eleven novel D- null alleles were identified, including one allele with a single missense mutation (RHD*443C>G; p.Thr148Arg) and one allele with a single amino acid deletion (RHD*424_426del; p.Met142del), both located in the transmembrane region of the RhD protein. The RHD*721A>C (found in seven individuals) and RHD*884T>C variant alleles cause Del expression and for the novel RHD*[178A>C; 689G>T] allele we postulate a partial weak D expression. In a single pregnant woman with an initial weak D typing an additional novel RHD variant allele, RHD*492C>A, was detected, causing partial weak D expression. The phenotypes of the RHD*443C>G, RHD*492C>A and RHD*721A>C mutation were confirmed with a heterologous expression study.

Conclusions: Fifteen new RHD variant alleles were identified and we determine for the first time that a single amino acid change or deletion can cause the D- phenotype.

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RHD variant alleles in RhD- individuals

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Introduction

The D antigen of the Rh blood group system is one of the most immunogenic and complex blood group antigens.1,2 D- individuals lack the complete RhD protein, which explains its high immunogenicity.3 When a D- individual comes into contact with the D antigen via transfusion or pregnancy, anti-D can be produced.4,5 Anti-D can cause severe hemolytic transfusion reactions and/or severe hemolytic disease of the fetus and newborn.6,7 To prevent anti-D formation in D- individuals compatible D- red blood cells are transfused and anti-D prophylaxis is administrated to D- pregnant women.8,9 The Rh locus is highly polymorphic and many RHD variant alleles have been described.10,11 The Rh locus consists of the highly homologous RHD and RHCE genes located in a tail-to-tail configuration on chromosome 1.12 One group of Rh variant alleles, the RHD hybrid alleles, arose due to genetic recombination in which part of the RHD gene are replaced by RHCE

counterparts.13,14 Another group of RHD variant alleles carry one or multiple mutations in the RHD gene.13 RHD variant alleles can cause the complete absence of the RhD protein, weakened expression of the D antigen or so called partial expression in which the RhD protein lacks one or more D epitopes.10,11 To date, more than 50, so called, D- null alleles have been described that cause the D- phenotype due to nonsense mutations, to mutations leading to a frame shift, to mutations that disrupt a splice site or to large hybrid alleles (Web Resources). The RDH*Ψ allele with a nonsense mutation and the hybrid RHD*03N.01 allele are D- null alleles that frequently occur in the Black population; in 66 % and 15% of D- Black individuals respectively.15-17 All other D- null alleles are rare.17 Individuals with a weak D phenotype express the RhD protein in low quantities that is most often caused by mutations in the transmembrane regions of the RhD protein.18 Individuals with Del expression have an even lower amount of the RhD protein on their red blood cell membrane, which can only be detected with the very sensitive adsorption-elution technique.19 Del expression is most often caused by mutations that disrupt a splice site.11 Partial D expression, in which one or more D epitopes are lacking, is most often caused by hybrid alleles or due to mutations in the extracellular parts of the RhD protein.20 Often RHD variant alleles causing partial D expression also cause weakened expression of the D antigen, the so called partial weak RHD variant alleles.10,11 The distinction between the different variant alleles is of importance, because it is unlikely that individuals with weak D or Del expression produce alloanti-D, in contrast to individuals with partial D expression who are at risk of D immunization.21 The aim of our study was to determine the frequency of (known and novel) RHD variant alleles among Dutch D- pregnant women. Since July 2011, Dutch D- pregnant women are offered a quantitative fetal RHD genotyping assay to guide anti-D prophylaxis. This quantitative fetal RHD genotyping assay is performed on maternal plasma, which contains cell free DNA of the fetus.22,23 The far majority of cell free DNA is of maternal origin and therefore the presence of

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a maternal RHD variant allele will result in much stronger signals in the quantitative PCR assay than expected to arise from fetal DNA. In this paper we present the serological and genetic follow up of cases identified among almost 38,000 screened Dutch D- pregnant women.

Material and methods

Samples and fetal RHD genotyping assayBetween July 2011 and December 2012 37,764 Dutch D- pregnant women were tested in the 27th week of pregnancy for the presence of a D+ fetus using a quantitative fetal RHD

genotyping assay based on TaqMan chemistry. DNA was isolated from 1 mL maternal plasma using a DNA isolation kit (DNA and Viral NA Large Volume Kit; Roche Holding AG) on a MagNa Pure 96 Instrument (Roche) via manufacturer’s protocol. The quantitative fetal RHD genotyping assay consist of two TaqMan tests, one targeting RHD exon 5 and one targeting RHD exon 7, which are performed in triplicate. This quantitative fetal RHD genotyping assay is extensively described by Scheffer et al.22 When at least two of the three Ct values of both assays were below 32 a maternal variant allele was suspected. When at least two of the three Ct values of exon 7 was below 32, while Ct values of exon 5 were above 32 a RHD*Ψ or RHD*06 maternal variant allele was suspected. In both cases material was stored and additional genotyping and extended serology was performed to determine whether a variant allele and which variant allele was present. Statistical analysis was performed using a 95% confidence interval [95% CI].

Serology Standard serology to determine the D phenotype in a pregnant woman was performed using two anti-D antibodies. A monoclonal anti-D reagent (immunoglobulin [Ig]M clone RUM-1, Sanquin Reagents) and a monoclonal blend reagent (IgM clone TM28 and IgG clone MS26, Sanquin Reagents) were used in a method with an immediate spin at room temperature. In all samples in which no agglutination was detected an indirect antiglobulin using the monoclonal blend reagent was performed. Plasmas of all women negative in this serological assay were tested in the quantitative fetal RHD genotyping assay. In women in whom a maternal variant allele was suspected, because Ct values were below 32 in this assay, a second serological test was performed. This second serological test consists of three monoclonal IgM anti-D (RUM-1, BS226 (Bio-Rad Laboratories B.V.) and ESD1-M (ALBA Bioscience)) that are tested in a method with an immediate spin at room temperature and in a method with a spin after 15 minutes incubation at room temperature and three monoclonal blend reagents (IgM clone TH28 and IgG clone MS26 (Sanquin reagents and ImmucorGamma) and IgM clone D7B8, IgG clone H112196 and IgG clone LORIFA (Ortho)), one monoclonal IgG (5C8) and a polyclonal IgG reagent (Bio-Rad Laboratories B.V.) that were tested in a method with an immediate spin at room temperature, in a method with 15 minutes incubation at 37ºC and a spin and/or in the indirect antiglobulin test. When also in this second test the D- phenotype was determined

Chap

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RHD variant alleles in RhD- individuals

83

and when the presence of the RHD*Ψ allele was excluded, absorption-elution was performed using the Gamma ELU-KIT II (ImmucorGamma) via manufactures’ protocol using a polyclonal anti-D (Bio-Rad Laboratories B.V.) to detect the very weak expression of a Del variant allele. If in the second serological test a partial D variant was suspected other than the DVI variant, a third serological test was performed using an in-house RhD typing kit consisting of eleven monoclonal IgG antibodies that target all epitopes of the RhD protein and/or the Extended Partial RhD Typing Set of Bio-Rad Laboratories.

RH-MLPAMaternal DNA was isolated from white blood cells using a DNA extraction kit (QIAamp DNA Blood Mini Kit. The copy number of the RHD gene and the presence of RHD variant alleles in the pregnant women, DNA samples were analyzed with the RH-Multiplex Ligation-dependent Probe Amplification (RH-MLPA) assay (mix p401, p402 and p403, MRC-Holland). If indicated, the RHCE-MLPA was performed.24 Furthermore, one cases was tested with seven new MLPA probe combinations, targeting the c.-698T, c.123A, c.149-4875A, c.149-882G, c.244T, c.335+2838C and c.1112G positions of RHD and RHCE, developed to determine the combined RHD and RHCE copy numbers of the 5’ UTR, exon 1, 2 and 8 and intron 1 and 2. The MLPA reaction was performed via manufacturer’s protocol on a Veriti Thermocycler (Applied Biosystems). In short, 5 μL containing 50-100 ng of DNA was denatured and 1.5 μL probe mix and 1.5 μL SALSA MLPA dilution buffer were added. After 16-20 hours of hybridization of the probe combinations to genomic DNA at 60°C, 1 μL SALSA ligase-65, 1.5 μL SALSA ligase buffer A and 1.5 μL SALSA ligase buffer B were added and incubated for 15 minutes at 54°C. A polymerase chain reaction [PCR] was performed on the complete ligation sample by adding 2 μL universal primers and 0.5 μL SALSA polymerase. PCR conditions were: 5 minutes at 72°C, 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C and 1 minute at 72°C, followed by 20 minutes at 72°C. A mixture of 1.5 μL MLPA sample, 8.5 μL Hi-DiT Formamide (Applied Biosystems) and 0.5 μL GeneScan 500-Liz Size Standard (Applied Biosystems) was analyzed on a 3130 Genetic Analyzer (Applied Biosystems). Data analysis was performed using Genemarker software version 1.85 (Softgenetics).

DNA sequencingIn some cases all exons and intron boundaries of RHD were sequenced. If indicated also the promoter region of RHD was sequenced (hg19, chr.1:g.25597899_25598887). The primers were synthesized by Eurogentec. The PCR was performed on a Veriti thermocycler in a total volume of 20 μL, containing 50-150 ng DNA, 10 μL of 2x GeneAmp Fast PCR Master Mix (Applied Biosystems), 0.5 μM forward and reverse primer. PCR conditions were: 10 seconds at 95°C, 35 cycles of 10 seconds at 95°C and a specific annealing/elongation temperature and time for each primer set ranging from 61 to 68ºC, followed by 1 minute at 72°C. PCR products were

Chapter 4

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purified using ExoSAP-IT (GE Healthcare), according to manufacturer’s protocol. The sequence reaction was performed on a Veriti thermocycler (Applied Biosystems) in a total volume of 20 μL, containing 1 μL of purified PCR product, 1 μL 2,5x BigDye Terminator v1.1 Cycle (Applied Biosystems), 3.5 μl 5x BigDye Terminator Buffer (Applied Biosystems) and 0.25 μM forward or reverse primer. Sequence conditions: 25 cycles of 15 seconds at 95°C, 10 seconds at 50°C and 4 minutes at 60°C. Sequence products were analyzed on a 3130 Genetic Analyzer (Applied Biosystems).

Heterologous expression systemAs a wild-type RHD construct the RHD coding sequence flanked by a BAMHI and NOTI digestion site was ordered at Invitrogen (Breda, The Netherlands) and cloned into the pHeftig vector, encoding GFP as a transduction control. The RHD*443G, RHD*492A, RHD*721C or RHD*1154C mutations were mutated into the wild-type RHD construct using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent) via manufactures’ protocol. K562 cells were lentivirally transfected with the RHD wild type construct, different variant constructs and a mock construct. Forty-eight hours after transduction, cells were harvested and screened for RhD expression by flow cytometry using nine monoclonal IgG anti-D of the ALBAclone Advanced partial RhD typing kit (ALBA Bioscience), namely LHM169/81, LHM76/59, LHM76/55, LHM50/28, LHM169/80, LHM57/17, LHM76/58, LHM59/19 and LHM77/664 and an additional IgG anti-Rh29 that can detect very weak D expression levels (BRIC69). Data analysis was performed with FlowJo Version 8 software (TreeStar).

Results

In 0.96% of the Dutch D- pregnant women a variant RHD allele is presentAll Dutch pregnant women are typed for their RhD status using a monoclonal IgM and monoclonal blend anti-D. Since July 2011, women who are typed D- in this initial test are offered a quantitative fetal RHD genotyping assay to test the RHD status of their fetus to determine whether they need to receive anti-D prophylaxis. The quantitative fetal RHD

genotyping assay is performed on DNA isolated from maternal plasma and targets exon 5 and 7 of RHD. Because cell free fetal DNA is present in an overwhelming background of maternal cell free DNA, maternal variant alleles disturb the quantitative fetal RHD genotyping assay. Of note, the frequently occurring RHD*03N.01 D- null allele is not amplified in this RHD

genotyping assay and is therefore not detected. To determine the range of Ct values that corresponds with a maternal allele, the fetal RHD genotyping assay was performed on 1000 D+ pregnant women, none of the women had Ct values of both exon 5 and 7 above 32 (data not shown). Hence, the cut-off value in which a maternal variant allele was suspected was set at a Ct value of below 32.

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RHD variant alleles in RhD- individuals

85

The quantitative fetal RHD genotyping assay was performed on a total of 37,764 D- pregnant women between 2011 and 2012. In 497 (1.3%) women a maternal variant allele was suspected, because Ct values of one or both exons were below 32. In 314 of the 497 women genetic follow-up of maternal variant alleles was performed on DNA isolated from white blood cells. In 129 of the 497 women no material was stored and no follow-up could be performed. Furthermore, in 54 cases in which a RHD*Ψ or RHD*06 allele was suspected (maternal amplification of only exon 7) no follow-up was performed, because in all other cases (n = 169) in which the presence of a RHD*Ψ or RHD*06 allele was suspected genotyping confirmed the presence of either one of these alleles. Except for one case that was homozygous for the RHD*01N.01 allele. In 39 (12.4%) of the 314 D- pregnant women in which genetic follow-up was performed no variant allele was identified, but the deletion of the RHD gene was detected (homozygous presence of the RHD*01N.01 allele). In these cases the Ct values arose from high fetal RHD

concentrations. In the remaining 275 women a variant allele was identified. The distribution of RHD alleles in cases in which follow-up was performed, was used to calculate the distribution of RHD alleles in cases in which no follow-up was performed (Supplementary Table S1). We estimate that in ~86 of the 183 cases in which no follow-up is performed a RHD variant allele is present (Supplementary Table S1). In conclusion, in 0.96% (95% CI 0.86% - 1.05%) of the Dutch D- pregnant women a variant allele that has RHD exon 5 and/or 7 is present and 47% of the women with a variant allele carries the RHD*Ψ allele.

Women determined D- with standard serology carried 45 different variant alleles In the 262 cases of the 275 cases with a variant allele red blood cells were available for extensive serological follow-up. In 145 (55%) cases the initial D- phenotype was confirmed, however, in 42 (16%) cases a Del phenotype, in 68 (26%) cases a partial D and in seven (3%) cases a weak D phenotype was determined. In 227 of the 275 cases on whom genotyping were performed, the RH-MLPA genotyping assay identified a specific RHD variant allele. In three of the 227 cases, additional sequencing was performed, because the Del phenotype of these cases did not correspond with the described weak partial D phenotype of the RHD*09.03 allele (with the c.[602C>G; 667T>C; 819G>A] mutations) that was identified by the RH-MLPA. In all cases an additional c.919G>A mutation was detected and these three individuals carried the recently described RHD*[602C>G;

667T>C; 819G>A; 919G>A] allele that causes the Del phenotype.25 In the remaining 48 cases additional genotyping was performed. In one case the RH-MLPA identified a novel variant allele, RHD*[361T>A; 380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A], that consists of the mutations of both the RHD*03.03 and RHD*09.03 allele. Sequencing confirmed the presence of this novel variant allele and no additional mutations were detected. In three cases with the Del phenotype the RH-MLPA detected an allele in which RHD exon 10 was absent. In

Chapter 4

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these cases the RHD*(1-9) or the RHD*(1-9)-CE(10) allele can be present, because both alleles have been described to cause the Del phenotype.25-27 The RHCE-MLPA detected two copies of RHCE exon 10 and we therefore conclude that these cases carried the RHD*(1-9) allele. In another case in which a RHD-RHCE-RHD allele was suspected the RHCE-MLPA and seven newly developed MLPA probe combinations (targeting the combined RHD and RHCE copy number of the 5’UTR, exon 1, 2 and 8 and intron 1 and 2) were performed. Taking the results of all MLPA probe combinations together in combination with the D- phenotype and RhCcee phenotype of this case, we postulate that this case most likely carries the known RHD*D-CE(2-9)-D allele (which is described to be linked with RhCe expression30) and a novel allele in which RHD exon 1 is deleted (RHD*(2-10)) (Supplementary Figure 1). In 43 cases additional sequencing was performed because the RH-MLPA was either indicating the presence of a normal RHD gene (n = 38) or was not able to identify the specific variant allele (n = 5). In twenty cases twelve novel RHD variant alleles were identified (Table 1). In one of the cases with a novel variant allele we detected the heterozygous presence of the c.178C and c.1136T mutation and the homozygous presence of the c.689T mutation. We presume that in this case, with partial D expression, the known RHD*10.01 and a novel RHD*[178A>C; 689G>T] allele are present, instead of two novel alleles (RHD*689C>T and RHD*[178A>C; 689G>T; 1136C>T]). All novel alleles were detected in single cases, except for the RHD*721A>C, allele, which was detected in seven cases and the RHD*1074-1G>A allele, which was detected in three cases (Table 1).A total of 45 different RHD variant alleles, including fourteen novel RHD variant alleles, were identified in the 275 cases, as listed in Table 1. Remarkably, in two cases, both with the Del and normal RhCE expression (RhCcee phenotype), an apparently normal wild-type RHD allele without mutations was detected. Also no mutations were detected in the promoter region of the RHD gene.

Characterization of fifteen novel RHD variant allelesExtended serological typing was performed in the cases carrying one of the fourteen novel RHD variant alleles. Furthermore, we analyzed another case carrying a novel variant allele (RHD*492C>A encoding p.Asp164Glu) discovered in a pregnant woman who had weak D expression in the initial D typing (Table 2). Eleven of the fifteen novel RHD variant alleles were determined to cause the D- phenotype by absorption-elution: the RHD*1084C>T allele (encoding p.Gln362Ter) with a nonsense mutation, two alleles (RHD*125_125delAA and RHD*1174delA) with frame shift mutations and the allele (RHD*(2-10)) in which RHD exon 1 was deleted and four alleles with mutations that disrupt a splice site (RHD*335G>T, RHD*[634+1G>T, 1136C>T], RHD*1073+1G>T or RHD*1074-1G>A) (Table 2). The novel allele that contained mutations of both the RHD*03.03 and RHD*09.03 variant alleles also causes the D- phenotype (Table 2).

Chap

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RHD variant alleles in RhD- individuals

87

Table 1. RHD variant alleles detected in 267 women determined D- with standard serology

RhD phenotype RHD allele 1 RHD allele 2Number of cases positive for genotype

D-

RHD*Ψ RHD*01N.01 102

RHD*Ψ RHD*01N.03 1

RHD*Ψ RHD*03N.01 13

RHD*Ψ RHD*Ψ 6

RHD*660delG RHD*01N.01 1

RHD*922G>T RHD*01N.01 2

RHD*952C>T RHD*01N.01 3

RHD*01EL.05† RHD*01N.01 1

RHD*01EL.08† RHD*01N.01 3

RHD*01EL.09† RHD*01N.01 1

RHD*(2-10)§ RHD*01-CE(2-9)-D 1

RHD*125_126del§ RHD*01N.01 1

RHD*335G>T§ RHD*01N.01 1

RHD*424_426del§ RHD*01N.01 1

RHD*443C>G§ RHD*01N.01 1

RHD*[361T>A; 380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A]§

RHD*01N.01 1

RHD*[634+1T;1136T]§ RHD*01N.01 1

RHD*1073+1G>T§ RHD*01N.01 1

RHD*1074-1G>A§ RHD*01N.01 3

RHD*1084C>T§ RHD*01N.01 1

RHD*1174del§ RHD*01N.01 1

Partial RhD RHD*04.02 RHD*01N.01 1

RHD*05.07 RHD*01N.01 1

RHD*06.01 RHD*01N.01 6

RHD*06.02 RHD*01N.01 40

RHD*06.02 RHD*03N.01 1

RHD*09.01 RHD*01N.01 2

RHD*09.01 RHD*03N.01 1

RHD*10.02 RHD*01N.01 2

RHD*11 RHD*01N.01 9

RHD*15 RHD*01N.01 12

RHD*17.02 RHD*01N.01 11

RHD*[178C; 689T]§ RHD*10.01 1

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Table 1. (Continued)

RhD phenotype RHD allele 1 RHD allele 2Number of cases positive for genotype

Del RHD*01EL.01 RHD*01N.01 9

RHD*01N.22‡ RHD*01N.01 1

RHD*[602C>G; 667T>G; 819G>A; 919G>A]

RHD*01N.01 2

RHD*[602C>G; 667T>G; 819G>A; 919G>A]

RHD*[602C>G; 667T>G; 819G>A; 919G>A]

1

RHD*93_94insT RHD*01N.01 6

RHD*(1-9) RHD*01N.01 2

RHD*(1-9) RHD*Ψ 1

RHD*1252_1253insT RHD*01N.01 1

RHD*721A>C§ RHD*01N.01 7

RHD*884T>C§ RHD*01N.01 1

RHD*01 RHD*01N.01 2

Weak RhD

RHD*01W.01 RHD*01N.01 3

RHD*01W.02 RHD*01N.01 3

RHD*01W.05 RHD*01N.01 1

RHD*01W.22 RHD*01N.01 1

RHD*01W.38 RHD*01N.01 1

† In literature it has been described that the RHD*01EL.05, RHD*01EL.08 and RHD*01EL.09 alleles cause the Del phenotype, we detected, however, in all cases carrying either one of these alleles the D- phenotype‡ In literature it has been described that the RHD*01N.22 allele cause the D- phenotype, we detected, however, the Del phenotype in the case positive for this allele§ Novel variant allele

All mutations of this variant allele (c.[361T>A; 380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A]) are located in the putative transmembrane or intra-cellular parts of the RhD protein (Figure 1). Unexpectedly, two novel variant alleles that caused the D- phenotype contained mutations that resulted in a single amino acid substitution or a single amino acid deletion in the transmembrane region of the RhD protein (Figure 1): the RHD*443C>G (encoding p.Thr148Arg) and RHD*424_426del (encoding p.Met142del) alleles (Table 2). The RHD*443C>G allele was already described by us in a single individual in a previous study24 and in this study we confirm the D- phenotype of the RHD*443C>G allele in another individual.Two novel alleles cause partial D expression, RHD*[178A>C; 689G>T] (encoding p.[Ile60Leu; Ser230Ile]) and RHD*492C>A (encoding p.Asp164Glu) with mutations in the fourth or third extracellular loop of the RhD protein, respectively (Figure 1). We were unable to determine the exact phenotype for the RHD*[178A>C; 689G>T] allele, because the case positive for this allele carried another variant allele, the RHD*10.01 allele. The epitope pattern in this case,

Chap

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4

RHD variant alleles in RhD- individuals

89

Tabl

e 2.

Fift

een

nove

l var

iant

alle

les

Alle

le n

ame

Nuc

leot

ide

Chan

ges†

Exon

(In

tron

)Pr

otei

n ch

ange

s‡Po

sitio

n in

RhD

pr

otei

nIn

itial

se

rolo

gyEx

tend

ed

sero

logy

Ads

orpt

ion-

Elut

ion

RhD

ph

enot

ype

Link

ed

RHCE

ge

noty

pe

RHD

*108

4C>

Tc.

1084

C>

T8

p.G

ln36

2Ter

N

egat

ive

Neg

ativ

eN

egat

ive

D-

RHC

E*02

RHD

*125

_126

del

c.12

5_12

6del

AA

1p.

Lys4

2fs

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

02

RHD

*117

4del

c.11

74de

lA9

p.Ile

392f

sN

egat

ive

Neg

ativ

eN

egat

ive

D-

RHC

E*02

RHD

*(2-

10)

c.1-

?_14

8+?d

el1

p.0?

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

01

RHD

*335

G>

Tc.

335G

>T

2r.s

pl?

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

01

RHD

*[63

4+1G

>T;

11

36T]

c.[6

34+

1G>

T;

1136

C>

T](4

) and

8r.s

pl?

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

01

RHD

*107

3+1G

>T

c.10

73+

1G>

T(7

)r.s

pl?

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

03

RHD

*107

4-1G

>A

c.10

74-1

G>

A(7

)r.s

pl?

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

02

RHD

*[36

1T>

A;

380T

>C;

383

A>G

; 45

5A>

C; 6

02C>

G;

667T

>G

; 819

G>

A]

c.[3

61T>

A; 3

80T>

C;

383A

>G

; 455

A>

C;

602C

>G

; 667

T>G

; 81

9G>

A]

3, 4

, 5

and

6

p.[L

eu12

1Met

; Val

127A

la;

Asp

128G

ly; A

sn15

2Thr

; Th

r201

Arg

, Phe

223L

eu;

Ala

273A

la]

6 m

utat

ions

in

tran

smem

bran

e re

gion

an

d on

e m

utat

ion

in

intr

acel

lula

ir re

gion

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

01

RHD

*443

C>G

c.44

3C>

G4

p.Th

r148

Arg

Tran

smem

bran

eN

egat

ive

Neg

ativ

eN

egat

ive

D-

RHC

E*02

RHD

*424

_426

del

c.42

4_42

6del

ATG

3p.

Met

142d

elTr

ansm

embr

ane

Neg

ativ

eN

egat

ive

Neg

ativ

eD

-RH

CE*

02

RHD

*721

A>C

c.72

1A>

C5

p.Th

r241

Pro

Tran

smem

bran

eN

egat

ive

Neg

ativ

ePo

sitiv

eD

elRH

CE*

01

RHD

*884

T>C

c.88

4T>

C6

p.M

et29

5Thr

Tran

smem

bran

eN

egat

ive

Neg

ativ

ePo

sitiv

eD

elRH

CE*

02

RHD

*[17

8A>

C;

689G

>T]

c.[1

78A

>C

; 689

G>

T]2

and

5p.

[Ile6

0Leu

; Ser

230I

le]

Tran

smem

bran

e an

d fo

urth

ext

race

llula

ir lo

opN

egat

ive

Part

ial§

-

Part

ial

wea

k D

RHC

E*01

RHD

*492

C>A

c.49

2C>

A4

p.A

sp16

4Glu

Third

ext

race

llula

ir lo

opW

eak

Part

ial

-Pa

rtia

l w

eak

DRH

CE*

02

† Po

sitio

n as

cou

nted

from

ATG

tran

slat

ion

star

t site

; hom

o =

hom

ozyg

ous,

hete

ro =

het

eroz

ygou

s‡

Posi

tion

as c

ount

ed fr

om M

et tr

ansl

atio

n st

art s

ite§

The

case

pos

itive

for t

his

varia

nt a

llele

car

ried

a se

cond

var

iant

alle

le R

HD

*10.

01 th

at c

ause

s pa

rtia

l D e

xpre

ssio

n, th

eref

ore

we

cann

ot e

xclu

de th

at th

is v

aria

nt a

llele

can

als

o ca

use

the

D- p

heno

type

Chapter 4

90

LegendLegendLegendLegend

Single amino acid changes of the RHD*424_426del allele encoding p.Met142del, the RHD*443C>G allele encoding p.Thr148Arg or the

RHD*1084C>T allele encoding p.Gln362Ter

Amino acid changes of the RHD*[361T>A; 380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A] allele encoding p.[Leu121Met; Val127Ala;

Asp128Gly; Asn152Thr; Thr201Arg, Phe223Leu; Ala273Ala]

Single amino acid changes of the RHD*721A>C allele encoding p.Thr241Proor the RHD*884T>C allele encoding p.Met295Thr

Single amino acid change of the RHD*492C>A allele encoding p.Asp164Glu

Amino acid changes of the RHD*[178C; 689T] allele encoding p.[Ile60Leu; Ser230Ile]

Extracellular region32323232 40404040

74747474 77777777

98989898 109109109109

133133133133 136136136136

154154154154 167167167167

188188188188 204204204204

230230230230 239239239239

261261261261 266266266266

285285285285 293293293293

318318318318 324324324324

347347347347 358358358358

391391391391

CCCC

IIIIIIII

NNNN

7777

Transmembrane region

Extracellular region

Intracellular region

Figure 1. The position of amino acid changes in the RhD protein present in seven different RHD variant alleles. Schematic representation of a two dimensional model of the RhD protein. Each dot represent an amino acid, dots within the black lines indicate putative membrane regions of RhD with residue numbers. Colored dots represent position of amino acids that are mutated in the RHD*424_426del, RHD*443C>G, RHD*1084C>T, RHD*[361T>A; 380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A], RHD*721A>C, RHD*884T>C, RHD*492C>A or RHD*[178A>C; 689G>T] allele.

loss of epitopes 1 (LHM70), 5 (rD7C2) and 8 (HIMA-36), corresponds to the epitope pattern described for the RHD*10.01 allele.28 We, however, postulate that for both variant alleles of this case the phenotype is caused by the c.689G>T mutation. Because, the additional mutation in the RHD*10.01 allele (c.1136C>T) has no effect on RhD expression in carriers of the RHD*10.00 allele28 and we assume that the additional mutation of the novel allele (c.178A>C) present in the transmembrane region of the RhD protein (Figure 1) also has no effect on RhD expression. The RHD*492C>A allele gave partial D expression, epitope 5 (rD7C2) and part of epitope 8 (HIMA-36 was negative, while LHM76/58 was positive) were not detected, while all other epitopes were normal positive (Table 3). The three tested IgM anti-D were unable to agglutinate red blood cells with c.492C>A mutation (Table 3) indicating that this variant allele has next to partial D also weakened D expression. Two novel alleles caused the Del

phenotype, the RHD*721A>C allele (encoding p.Thr241Pro) and RHD*884T>C allele (encoding p.Met295Thr), both with a single mutation in the putative transmembrane region of the RhD protein (Figure 1).

Chap

ter

4

RHD variant alleles in RhD- individuals

91

Tabl

e 3.

D-e

pito

pe e

xpre

ssio

n in

the

RHD

*443

G, R

HD

*492

A, R

HD

*[17

8C;6

89T]

, RH

D10

.01,

RH

D*7

21A,

RH

D*1

154A

and

RH

D*0

1 al

lele

s

RHD

*[17

8C;

689T

] and

RH

D*1

0.01

RHD

*10.

01†

RHD

*443

GRH

D*4

92A

RHD

*721

ARH

D*1

154A

RHD

*01

Mon

oclo

nal

antib

ody

RhD

ep

itope

Red

bloo

d ce

llsRe

d bl

ood

cells

Red

bloo

d ce

llsTr

ansd

uced

K5

62Re

d bl

ood

cells

Tran

sduc

ed

K562

Red

bloo

d ce

llsTr

ansd

uced

K5

62Tr

ansd

uced

K5

62Re

d bl

ood

cells

Tran

sduc

ed

K562

RT37

ºCIA

TIA

TRT

37ºC

IAT

RT

37ºC

IAT

RT

37ºC

IAT

RT37

ºCIA

T

LHM

701

-

-

-

+

3

LHM

169/

811

+-

-

4+

-

+/-

4+

5C8

2

+

-

3

-

3

LHM

76/5

93

+

-

+

+

/-+

/-

+LH

M76

/55

3

++

?

4

+

+/-

+/-

4

+AU

B-2F

7/Fi

ss5

-

rD7C

25

-

-

-

2

+LH

M50

/28

6/7

+

-

4+

-

+/-

4

+LH

M16

9/80

6/7

+

+

-

4+

+

/-+

/-

4+

LHM

57/1

76/

7

+

-

4

+

-+

/-

4+

LOS1

6/7

+

-

4

4

HIR

O-5

6/7

+

-

4

4

LHM

76/5

88

++

-

4+

-

+/-

4+

HIM

A-3

68

-

-

-

3

LHM

59/1

98.

2

+

-

-

-+

/-

+LH

M77

/64

9

+

+

-

4

+

-+

/-

+M

S26

9

++

3

Bl

end

anti-

D

(MS2

6 +

TM

28)

6/7,

9-

--

-

--

-

-3

-

--

44

4

RUM

-1-

-

--

-

--

--

4

4ES

D1-

M

--

--

--

--

4

4

BR

IC69

-

+

+/-

+

+Po

lycl

onal

an

ti-D

+

-

--

2

23

-

--

23

3

† ep

itope

pat

tern

des

crib

ed b

y W

agne

r et a

l.28

Chapter 4

92

RHD*443C>G RHD*492C>A

Negative non-transduced control

Positive wild type RHD*01 control

Specific RHD variant allele

Co

un

tC

ou

nt

Co

un

tC

ou

nt

Fluorescence iFluorescence iFluorescence iFluorescence intensity ntensity ntensity ntensity (arbitrary units)(arbitrary units)(arbitrary units)(arbitrary units)

RHD*721A>C RHD*01W.02

Figure 2. RhD expression levels of the RHD*443C>G, RHD*492C>A and RHD*721A>C variant alleles in a heterologous expression assay. Overlay plots of the fluorescence intensity representative for the RhD expression levels, of K562 cells transfected with constructs containing the RHD*443C>G, RHD*492C>A or RHD*721A>C cDNA (dark gray line with tinted area) with the RHD*01W.02 (light gray dotted line) or wild type RHD*01 cDNA (black line). The level of expression of the transporter (GFP) of RHD*443C>G, RHD*492C>A, RHD*721A>C and RHD*01W.02 compared to the wild-type RHD*01 control were, respectively, 31%, 96%, 91% and 115%. The RHD*01W.02 sensitivity control showed weakened RhD expression levels compared to the RHD*01 wild type allele. The RHD*443C>G allele had completely no RhD expression, the RHD*492C>A allele had exactly the same RhD expression level compared to the wild-type RHD*01 allele and the RHD*721A>C showed weakened RhD expression, even weaker compared to the RHD*01W.02 allele.

Expression of three variant alleles in a heterologous expression system It is relevant to correctly determine the phenotype produced by of a novel RHD variant allele, because carriers of alleles causing the partial D and D- phenotype can be immunized to the D-antigen, while this is unlikely for carriers of alleles causing the Del or weak D phenotype. Out of the fifteen novel variant alleles the phenotype could be assured for eight D- null alleles,

Chap

ter

4

RHD variant alleles in RhD- individuals

93

because these alleles contained a nonsense mutation, contained a frame shift mutation with a premature stopcodon, missed the complete exon 1 or contained mutations that affected a splice site. So far, of the remaining seven alleles the RHD*443C>G, RHD*721A>C and RHD*492C>A allele were examined in a heterologous expression system in K562 to confirm the phenotype determined on red blood cells. The wild-type RHD*01 allele and the RHD*01W.02 allele were tested as controls and indeed both were positive with all nine monoclonal IgG anti-D (Table 3). The RHD*01W.02 allele showed, however, lower expression levels compared to the RHD*01

allele (Figure 2). For all three RHD variant alleles, RHD*443C>G, RHD*721A>C and RHD*492C>A, the expression on the transduced K562 was similar to the phenotype determined on red blood cells. The RHD*443C>G allele was determined to give the D- phenotype with all anti-D (Table 3 and Figure 2). The RHD*721A>C allele showed very weak D expression levels, lower compared to the RHD*01W.02 allele (Figure 2). Furthermore, only a few anti-D (LHM76/59, LHM76/55 and LHM169/80) were detected very weakly positive (Table 3). The RHD*492C>A

allele showed normal RhD expression levels, the same as the RHD*01 allele (Figure 2), for all anti-D except LHM59/19 targeting epitope 8.2, which was completely negative (Table 3).

Discussion

In the present study we show that in ~1% of the Dutch D- pregnant women a variant allele is present that contains RHD exon 5 and/or exon 7. Genetic follow-up determined that almost half of the women with a variant allele carried the RHD*Ψ variant allele. In total we identified 45 different variant alleles, including fourteen novel RHD variant alleles. Overall serological follow-up detected in 55% of the women the D- phenotype, in 26% of the women partial D expression, in 16% of the women Del expression and in 3% of the women very weak D expression. Interestingly, in two cases, with a wild-type RHD*01 allele and without any mutation in the intron boundaries or in the promoter region of the RHD gene, the Del and RhCcee phenotype was detected. The RhCe expression was normal in these two cases. Flegel et al.29 also described a single case without mutations in the RHD exons and intron boundaries with the Del and a normal RhCe phenotype. Possibly, in these cases a gene that is required for membrane expression of the RhD protein is mutated. In six cases carrying four variant alleles RHD*01EL.05 (n = 1), RHD*01EL.08 (n = 3), RHD*01EL.09 (n = 1) and the RHD*01N.22 (n = 1) the phenotype that was detected in this study deviated from the phenotype as reported for these variant alleles. Several Del variant alleles, including the RHD*01EL.08 and RHD*01EL.09, have been described to give both the D- and Del phenotype11,29,30 and the RHD*01EL.05 allele was originally described in single case with the Del phenotype31. It is assumed that Del expression arises from a small degree of normal splicing despite the mutation at the splice site consensus.31,32 It may be that the level of expression in certain Del variant alleles is too low to be detected by the adsorption-elution technique. Yet, allo-immunization has occurred in individuals carrying the RHD*01EL.08 allele33 or the

Chapter 4

94

RHD*01EL.05 allele10, hence individuals positive for the RHD*01.05 and RHD*01EL.08 allele are considered to be at risk of D immunization. Finally, in a single case carrying the RHD*01N.22

allele we detected the Del phenotype, the same phenotype as was detected by Flegel et al.29, while in the initial case with this genotype the D- phenotype was discribed34. The RHD*01N.22

allele encodes a truncated RhD protein that lacks a small part of the C-terminal intracellular tail. Because the nonsense mutation is at the end of the RHD protein, possibly some of this truncated RhD protein is able to incorporate into the membrane. For instance, the Del

phenotype is also detected in individuals carrying the RHD*(1-9) allele, including three cases carrying the RHD*(1-9) allele of this study, that encodes a truncated RhD protein only nine amino acids longer than the truncated RhD protein encoded by the RHD*01N.22 allele.25

We identified fourteen novel alleles (eleven D- null, one partial weak and two Del alleles) in our cohort of D- pregnant women and one additional novel allele was identified in a pregnant woman with weak D expression in the initial serological typing. The D- phenotype was determined for a novel allele with a nonsense mutation (RHD*1084C>T), two novel alleles with frame shift mutations (RHD*125_125delAA or RHD*1174delA), four alleles with mutations that disrupt a splice site (RHD*335G>T, RHD*[634+1G>T, 1136C>T], RHD*1073+1G>T or RHD*1074-

1G>A) and one allele with the entire deletion of exon 1 (RHD*(2-10)). The presence of this last allele could not be unambiguously proven, because of the presence of another RHD variant allele in this case. The D- phenotype was also determined in a case positive for a variant allele that contained mutations of both the RHD*03.03 and RHD*09.03 variant alleles (RHD*[361T>A;

380T>C; 383A>G; 455A>C; 602C>G; 667T>G; 819G>A]). Because, the RHD*09.03 allele causes only a moderate weakening of the RhD expression and the RHD*03.03 allele has completely no effect on RhD expression levels, it is unexpected that the combination of these mutations completely abolishes RhD expression levels. Interestingly, one allele with a single missense mutation (RHD*443C>G encoding p.Thr148Arg) and one allele with the deletion of a single amino acid (RHD*424_426del encoding p.Met142del) also cause the D- phenotype. For the RHD*443G allele the D- phenotype was confirmed in a heterologous expression study in K562. Both alleles have mutations in the fifth putative transmembrane region of the RhD protein. Other RHD variant alleles with mutations in this region, drastically diminish D expression, for instance the RHD*01EL.07 (encoding p.Ala137Glu) and RHD*01EL.12 (encoding p.Leu153Pro) alleles that cause Del expression27,29, these alleles, however, do not completely abolish D expression as was detected in the RHD*443G and RHD*424_426del allele. Two novel variant alleles (RHD*[178A>C; 689G>T] encoding p.[Ile60Leu;Ser230Ile] and RHD*492A encoding p.Asp164Glu) caused partial weak D expression. Because the case positive for the RHD*[178A>C; 689G>T] allele also carried the RHD*10.01 allele, we were unable to determine the exact phenotype of this variant allele neither to unambiguously demonstrate the presence of this novel allele. We postulate that the c.689T mutation (p.Ser230Ile) which is present in both variant alleles of this case is responsible for the phenotype, hence a weak

Chap

ter

4

RHD variant alleles in RhD- individuals

95

partial expression in which epitope 1, 5 and 8 are absent. The RHD*492C>A allele causes weak partial D expression on red blood cells and using the heterologous expression system in which epitope 5 and part of epitope 8 were absent. The novel RHD*721A>C allele (encoding p.Thr241Pro) and RHD*884T>C (encoding p.Met295Thr) with a single missense mutation in the transmembrane region of the RhD protein cause the Del phenotype. The phenotype of the RHD*721A>C allele was confirmed using the heterologous expression system. Interestingly, this variant allele was detected in seven cases, whereas all other novel alleles, except the RHD*1074-1G>A allele, were detected in only a single case. This allele was not detected in the studies of Flegel et al.29, Polin et al35 and Orzinska et al.36 performed in Germany, Austria and Poland, respectively, indicating that this allele is specific for the Dutch population. All women positive for the RHD*721A>C allele had common Dutch surnames

In conclusion, 0.96% of the Dutch D- pregnant women carry a D variant allele harboring RHD exon 5 and/or exon 7. The far majority of pregnant women with a variant allele carry a D- null allele or partial D allele and need administration of anti-D prophylaxis to prevent anti-D immunization. Genotyping of this group of women has the advantage that the woman with weak D type 1, 2, 3 or 5 are recognized and can be regarded in the current and, possible next pregnancies and as blood donor as D+. Furthermore, this cohort of extensively typed D- women can be used to optimize RHD genotyping assays, because for correct prediction of the D phenotype via a genotyping assay it is essential that the most frequently occurring D- null alleles are identified.

Web Resources

Inventory of D- null alleles. http://www.uni-ulm.de/~fwagner/RH/RB2/P_RHDDnegative.htm. Accessed 20-08-2013

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96

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26. Silvy M, Chapel-Fernandes S, Callebaut I, Beley S, Durousseau C, Simon S, Lauroua P, Dubosc-Marchenay N, Babault C, Mouchet C, et al. Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture. Transfusion 2012;52(9):2020-9.

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30. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC.Genet. 2001;2:10.

31. Singleton BK, Green CA, KIMURA K, MINAMI A, Okubo Y, Daniels GL. Two new RHD mutations associated with the Del phenotype. Transf.Clin.Biol. 8[Suppl 1], 9S. 2001.

32. Liu HC, Eng HL, Yang YF, Wang YH, Lin KT, Wu HL, Lin TM. Aberrant RNA splicing in RHD 7-9 exons of DEL individuals in Taiwan: a mechanism study. Biochim.Biophys.Acta 2010;1800(6):565-73.

33. Kormoczi 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):1561-7.

34. Gassner C, Doescher A, Drnovsek TD, Rozman P, Eicher NI, Legler TJ, Lukin S, Garritsen H, Kleinrath T, Egger B, et al. Presence of RHD in serologically D-, C/E+ individuals: a European multicenter study. Transfusion 2005;45(4):527-38.

35. Polin H, Danzer M, Gaszner W, Broda D, St-Louis M, Proll J, Hofer K, Gabriel C. Identification of RHD alleles with the potential of anti-D immunization among seemingly D- blood donors in Upper Austria. Transfusion 2009;49(4):676-81.

36. Orzinska A, Guz K, Polin H, Pelc-Klopotowska M, Bednarz J, Gielezynska A, Sliwa B, Kowalewska M, Pawlowska E, Wlodarczyk B, et al. RHD variants in Polish blood donors routinely typed as D-. Transfusion 2013.

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Chap

ter

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RHD variant alleles in RhD- individuals

99

Supporting informationChapter 4

Chapter 4

100

Supp

lem

enta

ry T

able

S1.

Cal

cula

tion

of n

umbe

r of c

ases

with

a R

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the

tota

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764

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omen

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on 7

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l

Num

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Num

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Num

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var

iant

al

lel i

s pr

edic

ted

to b

e

Num

ber o

f cas

es

pred

icte

d to

be

posi

tive

for t

he

pres

ent

abse

ntRH

D*Ψ

al

lele

RHD

*06

alle

lepr

esen

tab

sent

RHD

*Ψ

alle

leRH

D*0

6 al

lele

<30

<30

9484

813

109.

60.

4

<30

30-3

110

109

1

0

<30

31-3

20

<30

>32

11

10

0

30-3

1<

303

33

01

0

30-3

130

-31

1310

73

3

2.1

0.9

30-3

131

-32

158

44

7

3.5

3.5

30-3

1>

321

10

1

0

31-3

2<

303

33

02

10

31-3

230

-31

21

10

1

1.0

0.0

31-3

231

-32

4616

115

30

1.9

28.1

31-3

2>

3270

80

8

620.

062

.0

>32

<30

184

127

126

193

3357

56.6

0.4

42.1

14.9

>32

30-3

145

3535

025

910

10.0

0.0

7.4

2.6

>32

31-3

210

74

31

33

1.7

1.3

Tota

l 

497

314

275

3912

246

183

8697

4918

Chap

ter

4

RHD variant alleles in RhD- individuals

101

Nu

cle

oti

de

po

siti

on

c.-698T

c.-132A or c.-132G

c.48C (targetting RhC and RhD)

c.123A

c.149-882G

c.149-4875A

c.149-20G (targetting RhC and RhD)

c.149-109nt insertion (targetting RhC)

c.203G (targetting RhC and RhD)

c.244T

c.307C (targetting Rhc)

c.335+2838C

c.380T or c.380C

c.455A

c.514A

c.602C or c.602G

c.667T

c.676G or c.676C

c.697G

c.787G or c.787A

c.800A

c.932A or c.932G

c.941G

c.989A or c.989C

c.1061C

c.1112G

c.1193A or 1193T

c.1357C

Exo

n o

r (i

ntr

on

)(2

)(3

)(4

)(5

)6

(6)

(7)

8(8

)9

(9)

10

MLP

A r

atio

fo

r R

HD

0,8

1,1

1,0

1,0

1,0

1,0

0,9

1,0

0,9

1,2

0,8

0,9

0,8

1,0

1,9

MLP

A r

atio

RH

CE

2,1

2,3

0,9

2,9

2,8

3,1

2,9

2,9

2,8

2,8

2,1

Co

mb

ine

d M

LPA

rat

io o

f R

HD

an

d R

HC

E 3

,01

,83

,23

,23

,62

,93

,04

,13

,53

,6

RH

D*(

2-10

)

RH

D*0

1-C

E(2-

9)-D

RH

CE*

01

RH

CE*

02

5'U

TR

17

(1)

23

45

Supplementary Figure S1. Schematic representation of MLPA results in a single case with two RHD variant alleles. The ratio of the MLPA, representing the copy number of the RHD, RHCE or combined RHD and RHCE genes, is translated in a model of the RHD and RHCE alleles. Exons of the RHD gene are depicted has black boxes and exons of the RHCE gene are depicted as open boxes. The combined copy number of RHD and RHCE is 3 for the 5’ UTR and exon 1 indicating that one of the RHD or RHCE alleles lacks the expression of the 5’ UTR and exon 1. Furthermore, the RHCE exon 3, 4, 5, 6, 7 and nine have a copy number of 3 indicating the presence of a RHD-RHCE-RHD hybrid allele. We postulate that in this case, with the Del and RhCcee phenotype, a novel variant allele RHD*(2-10) is present in which exon 1 is deleted linked with the RHCE*01 allele and the known RHD*01-CE(2-9)-D(10) allele linked with the RHCE*02 allele are present.