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1995 85: 829-832

der SchootBH Faas, S Simsek, PM Bleeker, MA Overbeeke, HT Cuijpers, AE von dem Borne and CE vanRh E/e genotyping by allele-specific primer amplification

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reserved.Copyright 2011 by The American Society of Hematology; all rights900, Washington DC 20036.weekly by the American Society of Hematology, 2021 L St, NW, SuiteBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published

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Rh E/e Genotyping by Allele-Specific Primer AmplificationBy B.H.W. Faas, S. Simsek, P.M.M. Bleeker, M.A.M. Overbeeke, H.Th.M. Cuijpers, A.E.G. Kr. von dem Borne,

and C.E. van der SchootIthas been hown that the hesus (Rh) lood group antigensare encodedbytwo homologous genes: the RhD gene andthe RhCcEe gene. TheRhCcEegene encodes different pep-tides: theRhC,cE, ande polypeptides. Only one ucleotidedifference has beenound between the alleles encoding heRh E and the Rhe antigen polypeptides.It is aC -+ G transi-tion at nucleotide position 676, which leads to an aminoacid substitution from proline to alanineinthe Rh e-carryingpolypeptide. Here we present an allele-specific rimer ampli-fication (ASPA) method to determine the Rh E and Rh egenotypes. In onepolymerasechainreaction, the senseprimer had a 3'-end nucleotide specific for the cytosine atposition 676 of the Rh E allele. in another reaction, a senseprimer was used with a 3'-end nucleotide specific for theguanine a t position 676 of the Rh e allele and the Rh D

HE RHESUS (Rh) blood group system is of clinicalinterest, because it is involved in hemolytic disease ofthe newborn (HDN), in hemolytic transfusion reactions, and

in autoimmune hemolytic anemia (AIHA). The Rh systemis complex; as many as 46 different antigens have beenserologically defined.'.' Among these antigens are those ofthe Rh D, C/c, and We series. The two highly homologousgenes encoding these antigens are localized on chromosomelp34.3-p36.1 and are inherited t~gether.~ne gene encodesthe Rh D antigen. The Rh D-negative phenotype is causedby the absence of the entire or at least part of the Rh Dgene5 rather than by an allele of the gene. The other gene,the Rh CcEe gene, encodes the polypeptides carrying the RhC/c as well as those carrying the Rh E/e polymorphisms.Recently, the cDNA structure of the Rh gene has been eluci-dated.6.7The Rh CcEe gene encodes different polypeptides,and there are several alleles. Alternative splicing probablyplays a role in the production of these polypeptides.' In thelimited number of donors tested so far, only one nucleotidedifference has been found between the alleles encoding thepolypeptide carrying the Rh E and the Rh e antigen.'.'' Thisdifference involves the nucleotide at position 676 of thecoding sequence6,' and leads to aproline to alanine substitu-tion in the allele encoding theRh e antigen.

Until recently, it was only possible to determine the Rhphenotype by serologic typing of red blood cells. This sero-logic approach can be inconclusive, eg, in the case of Rhphenotyping of fetuses and of patients who have recentlybeen transfused and who harbor a large quantity of donorred blood cells. In these cases, Rh genotyping is an option.Methods to determine the Rh D genotype on genomic DNAhave been de~cribed.~~""~s HDN, AIHA, and transfusionreactions are not only due to anti-Rh D antibodies but alsosometimes to anti-Rh We or anti-Rh C/c antibodies, it isimportant also to be able to determine the Rh We and RhC/c genotype in such cases. Recently, Hyland et a l l 4 appliedrestriction fragment length polymorphism (RFLP) patternson Southern blots for Rh genotyping. However, they founda 100% correlation for 102randomly selected blood donorsfor the Rh C, Rh e, and Rh D phenotypes, but only 94.8%for the Rh c and 94.3% for the Rh E phenotypes.

T

Blood, Vo l 85, No 3 (February 1). 1995:pp 829-832

gene, whereas the antisense primer had a 3'-end ucleotidespecific for the denine at position 87 of the Rh CcEe gene.We tested DNA samples from 158 normal donors (includingnon-Caucasian donors andonorswith rareRhphenotypes)in these assays. There was full concordancewiththe resultsof serologic Rh E/e phenotyping. Thus, we may concludethat the ASPA approach eads to a simple and reliablemethod to determine the Rh E/e genotype. This an be use-ful inRhlegenotypingof fetuses and/or incases inwhichno red blood cells are available for serotyping. Moreover,our results confirm the proposed association between thecytosine/guanine polymorphism at position676 and the RhEle phenotype.0 1995 by The American Societyof Hematology.

Here we describean Rh E/e genotyping method using apolymerase chain reaction (PCR) with two primer sets, withthe 3'-end nucleotides of one or both primers specific toamplify the Rh EorRh e allele [allele-specific primer ampli-fication (ASPA)]. We performed these two PCRs to deter-mine the Rh E and Rh e genotypes in DNA samples from158 white and non-white volunteer blood donors thathad been serologically phenotyped for Rh D, Rh We, andRh c/c.

MATERIALS AND METHODSRed blood cells from 158 mainly white donors were serologically

typed for the Rh C/c,=e,and D phenotypes (Table 1). Murinemonoclonal antibodies (Pelikloon anti" D, anti-RhE, anti-Rh e,anti-Rh C, and anti-Rh c; all gM, CLB, Amsterdam, The Nether-lands) as well as polyclonal human antibodies (anti-Rh D, anti-RhE, anti-Rh e, anti-Rh C, and anti-Rh c for the bromelin technique;CLB) wereused. High-molecular weight DNA was extracted fromthe eukocytes of these donors by standard methods described byCiulla etd.'5To performanRh E-specific ASPA, the ollowing two sets ofprimers were used: set A, primer R661csense primer: S'CCAAGT-GTCAACTCTC3', position 661 to 676 of the codingandprimer R768 (antisenseprimer:S'TGACCCTGAGATGGC-TGT3'. position 768 to 751); and set B: primer R487 (sense primer:S'ACAGACTACCACATGAAC3', position487 to 504) and primerR568 (antisense primer: S'GCT'rTGGCAGGCACCAGGCCAC3',

From the Central Laboratory of the Netherlands RedCrossBloodTransfusion Service and the Luboratory for Experimental and Clini-cal Immunology, University of Amsterdam; and the Department ofHaematology, Academic M edical Center, Amsterdam, The Nether-lands.Submitted May 23, 1994; accepted October 3, 1994.Address reprint requests to C.E . van der Schoot, MD, PhD, Cen-tral L aboratory of the Netherlands Red Cross Blood TransfusionService, 1066CXAmsterdam, The Netherlands.The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C.section 1734 solely toindicate this facr .0 1995 by The American Society of H ematology.0006-4971/95/8503-0$3.00/0

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830 FAAS ET AL

Table l.Serologically Predicted Phenotypes and E-Specific and e-Specific ASPA-Determined Genotypes of DNA Samples

E-Predicted No . of Donors Specific Specific

e-Serologic Type' Tested ASPASPA Genotype

CDelCDe[81CDe/ce [81CDe/cDE [91cDE1ce161cDe/ce [1cDE/cDEce/ce [l41CelcecE/ceCDecDE (Rhi')CDEICDECEICECDEICDeCDE/cDE

23( 5nonwhite)

33( 3nonwhite)171111

( 5nonwhite)8

3210

(1 nonwhite)6

(1 nonwhite)21121

+++++~

++++~

-+~

Numbers in brackets indicate donors for whom the predicted phe-notypes are not confirmed by family analysis.

position 568 to 547). To perform an Rh e-specific ASPA , set Bwas used in combination with set C: primer R661g (sense primer:S'CCAAGTGTCA ACTCTG3', position 661 to 676) and primerR801 (antisense primer: 5'CA TGCTGA TCTTCCT3', position 801to 787). All primers were synthesized on a DNA synthesizer (Ap-plied Biosystems model 392, Palo A lto, CA), and the primers R661 c,R661g, and R801 were purified with oligonucleotide purificationcartridges (Applied Biosystems, Foster City, CA ).The PCR was performed on 0.7 pg of genomic DNA template ina total volume of 50pL. The reaction mixture contained 75 ng ofeach primer, 0.2mrnol/L of each dNTP (Pharmacia, Uppsala, Swe-den), 2 U of Taq DNA polymerase in the appropriate buffer (Pro-mega, Madison, WI), 2.5 mmol/L MgC12 for the E-specific ASPAand 1.5mmol/L MgClz for the e-specific A SPA . A negative controlsample without DNA was always included. Thirty-five cycles ofamplification were performed in a thermal cycler (Perkin ElmerCetus model 480, Norwalk, C T), with denaturation for 1 minute at95"C, annealing for 1.5minutes at 62C for primer sets A and Band 1.5 minutes at49C for primer sets B and C, and extensionfor 2.5 minutes at 72C. The products were separated on a 10%polyacrylamide gel and visualized with ethidium-bromide staining.

RESULTSPrimers R661cfR768 (setA ),R487fR568 set B), andR661gR801 (set C) were designed to ampli fy specific re-gions of the Rh CcEe and Rh D genes. Some experimentswere performed to arrive at the optimal conditions to usethe primer sets. PCRs were performed with annealing tem-peratures of 46"C, 49"C, 55T , and 62C. MgC12 concentra-tions were varied using the optimal annealing temperature

(1.0 mmol/L , 1.5 rnmolk, 2.0 mmol/L , and 2.5 mmoVL;results not shown). The combination of primer sets A andB worked well in a large range of annealing temperatures;

between 46C and 62"C, bands were always obtained. How-ever, at lower temperatures more aspecific bands were seen.A higher concentration of MgCIZ resulted in stronger bands.The optimal conditions or theE-specific A SPA were anannealing temperature of 62C and 2.5 mmol/L M gCI2. Thecombination of primer setsB and C only gave good roductsat annealing temperatures of 46C and 49C. At higher tem-peratures, the e-specific band was lost, whereas the controlband was still visible. Changing the M gClz concentrationsresulted in the oss of the e-specificbandat 2.0 mmol/LMgC1,. T hus, the e-specif ic ASPA is optimal at49C anneal-ing temperature, using 1.5mmol/L M gCI 2.Set A amplifies a 108-base pair region specific for the RhE allele of the Rh CcEe gene (exon S ) because of the useof the 3'-end nucleotide of primer R661c, which is specificfor nucleotide 676of the Rh E allele. Set B amplifies a 94-

\,i

Fig 1. Schematic diagram of the ASPA for Rh E and Rh e genotypins. In each reaction, tw o primer sets are used: a control set thatam pl ie s a 94-bp region common to both the Rh CcEe gene and theRh D gene and a primer set specific for the Rh E or Rh e allele of theRh CcEe gene. IA) Schematic result of the Rh E-specific ASPA, inwhich primer set A (R661cIR768) is used to amplify an Rh E-specificregion of 108 bp. (B ) Schematic result of the Rh e-specific ASPA, inwhich primer set C (R66lglR801) is used to amplify an Rh e-specificregion of 141 bp (x =E or e).

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Rh /e GENOTYPING 83 1

1 2 3 4 5 6 Mn0 0 b p141 b p108 b p-9 4 b p- - 00 b pFig 2. Rh Ele genotyping by ASPA analysis. RhE- and Rh e-specific PCRs were performed using Rh E or Rh e allele-specific primers [set A(108 bp) and C (141 bp), respectively]. Lanes 1 and 2 DNA sample from a homozygous ccDEE donor, amplified with primer set C (lane 1) andprimer Set A (lane 2); lanes 3 and 4 DNA sample from a heterozygous CcDEe donor, amplified with primer set C (lane 3) and primer set A(lane 4); lanes 5 and 6 DNA sample from a homozygous ccdee donor, amplified with primer set C (lane 5) and primer set A (lane 6); lane M:marker DNA. In all lanes, primer set B, which amplifies a product of 94 bp, was used as a positive control.bp egion in exon 4 common to the Rh D gene and theRh CcEe gene. The 108-bp band and the 94-bp band werecoamplified in one PCR. In Rh E-negative DNA samples(ee). only the control fragment of 94 bp is found, whereasin Rh E-positive DNA samples (Ee or EE), the control frag-ment aswell asthe Rh E-specific 108-bp fragment are ampli-fied (Fig IA).

Set C amplifies a 141-bp region of the Rh CcEe genespecific to theRh e allele. Primer R661g hasa3'-end nucleo-tide (nucleotide 676) that enables the primer to bind to theRh D gene as well as to the Rh e allele of the Rh CcEegene, but not to the Rh E allele. Primer R801 hasa 3'-endnucleotide (at position 787) that is specific for theRh CcEegene but not the Rh D gene. When primer sets B and C areused in the ASPA assay, only the 94-bp control fragment isamplified in Rh e-negative DNA samples, while in Rh e-positive DNA samples two bands are produced: the 94-bpcontrol bandand the 141-bp Rh e-specific fragment (Fig1B).All of the l58 samples were serologically phenotyped forRh C/c, We, and D (Table l). Using the Rh E- and Rh e-specific ASPA, we typed all of the donors for their Rh E orRh e genotype. The results of the E-specific and e-specificASPA and the conclusions with regard to the genotype arelisted in Table 1 . There was complete agreement with theresults of the serologicRh We phenotyping. All of the sam-ples that were serologically typed asEE or Ee produced twobands when using primer sets A andB in the ASPA reaction,whereas all of the samples that were serologically typed asee onlyproduced the lower band in this reaction (Fig 2).This pattern was foundnot only in the combination of Ewith c, but also of E with C (see the rare CDE/CDE, CdEICdE and CDE/CDe phenotypes). All samples that were sero-logically typedasEe or ee produced two bands when primersets B and C were used in theASPA: the Rh e-specificband of 141 bp and the control band. All samples that wereserologically typed as EE only produced the control band(Fig2).

DISCUSSIONUntil recently, it was only possible to determine the RhC/c, Rh E/e, andRh D phenotypes serologically; but because

the messenger RNA sequences of the Rh CcEe and Rh Dgenes have become known," several methods have been de-

scribed to determine theRh D genotype at the genomic DNAIeveI.""3In the present study, weused woASPA reactions to

determine the Rh Ele genotype on genomic DNA. To ruleout false-negative reactions caused by insufficient quality ofthe reagents or failure of the reaction itself, we included aninternal control, primer set B, that always gives an amplifi-cation product irrespective of the phenotype. We performedthe reactions onDNA samples from 158 volunteer blooddonors with diverse serologically determined Rh CcDEephenotypes. The results of our Rh E and Rh e genotypingwere in full concordance with the results of the serologicphenotyping. By genotyping DNA samples from donors withrare alleles (CDE and CdE), it was shown that only the cEcombination, which is by far the most common, but also theCE combination could be amplified.

Recently, Hyland et all4used Msp I RFLP digestion pat-terns of the3' noncoding regions of the genes to determineRh Ele genotypes. For e they showed a 100% concordancebetween the results of serologic phenotyping and genotypingbased on RFLP patterns, but for E the concordance was only94.3%. The discrepancies they found between the results ofserologic phenotyping and molecular genotyping appearedto be associated with the cE allele in D-negative subjects.The six cE alleles in four D-negative donors whose DNAwas tested were all genotypedasce. No discrepancies wereseen when the cE allele occurred in ahaplotype with the Dgene. From these results, the investigators concluded thatthe cE allele is different in the presence or absence of the Dgene. However, because they were only analyzing noncodingregions, these results only show linkage between theM sp IRFLP and the Rh Ele phenotype. Moreover, no DNA fromeither D-positive or D-negative subjects with the CE allelewas typed. In theASPA method, we tested DNAof sixdonors containing the cE haplotype and never encounteredadiscrepancy. Furthermore, wealsotested some DNA sam-ples from rare phenotypes. These results were also in fullconcordance. For RFLP pattern analysis,a large quantity ofDNA is needed, whereas for this PCR-based method,a smallamount of genomic DNA is sufficient. This small amount-purified rom less than 0.5 mLof wholeblood or fromamniotic cells-makes genotyping samples from fetuses andfrom patients with severe transfusion problems feasible.

Although the results of our Rh Ele genotyping were in

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832 FAAS ET AL

full concordance with the results of the Rh Ele phenotyping,one must be aware of the fact that discrepancies might occurin rare cases, such as cases in which there is transmissionof silent alleles at the Rh locus. In these cases (eg, theRh,,,, and theD- phenotypes), theRh CcEe gene is presentwithout anygenomic rearrangementsormutations within thecoding region, but no Clc or Ele antigens can be detectedon the erythrocytes.I6

Our results obtained from this large group of donors con-firm the proposed association between the cytosineor gua-nine polymorphism at position676of the Rh CcEe gene andthe Rh E or Rh e phenotype, respectively. These findingsstrongly suggest that proline and alanine are involved in thespecific epitopes that are recognized by anti-Rh E and anti-Rh e antibodies, respectively.In conclusion, we present a simple and reliable PCR-basedmethod to determine the Rh Ele polymorphism on genomicDNA. This can be useful to determine the Rh E/e genotypein fetuses inan early phase of pregnancy and n recentlytransfused patients with large amounts of circulating donorcells.

ACKNOWLEDGMENTWe thank the staff of the Department of Blood Group Serologyfor help serotyping the blood samples and Dr C.P. Engelfriet forcomments on the manuscript.

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3. ChCrif-Zahar B, MattCi MG, Le Van Kim C, Bailly P, CartronJ-P, Colin Y: Localization of the human Rh blood group gene struc-ture to chromosome region lp34.3-lp36.1 by in situ hybridization.Hum Genet 86:398, 19914. ColinY, ChCrif-Zahar B, LeVan Kim C, Raynal V, Van HuffelV, Cartron J-P: Genetic basis of the RhD-positive and RhD-negativeblood group polymorphism as determined by Southern analysis.Blood 78:2747, 19915. SimsekS , Faas BHW, Bleeker PMM, Overbeeke MAM, Cuijp-ersHThM, van der Schoot CE, von dem Borne AEGKr: Rapid RhDgenotyping by PCR-based amplification of DNA. (submitted).6. Avent ND, Ridgwell K, Tanner MJA, Anstee DJ: cDNA clon-ing of a 30 kDa erythrocyte membrane protein associated with Rh(Rhesus)-blood-group-antigen expression. Biochem J 271 :821, 19907. ChCrif-Zahar B, Bloy C, Le Van Kim C, Blanchard D, BaillyP, Hermand P, SalmonC,Cartron J-P, Colin Y: Molecular cloningand protein structure of a human blood group Rh polypeptide. ProcNatl Acad Sci USA 87:6243, 19908. LeVanKim C, Chbrif-Zahar B, Raynal V, Mouro I, LopezM, Cartron J-P, Colin Y: Multiple Rh messenger RNA isoformsareproduced by alternative splicing. Blood 80:1074, 1992

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10. Simsek S , de Jong CAM, Bleeker PMM, Westers TM,GoldschmedingR, van der Schoot CE, Cuijpers HThM, von demBorne AEGKr: Sequence analysis of cDNA clones derived fromreticulocyte mRNAs coding for Rh polypeptides. Vox Sang 67:203,199411. Bennett PR, Le VanKim C, Colin Y, Warwick RM,PathFRC, ChCrif-Zahar B, Fisk NM,Cartron J-P: Prenatal determinationof fetal RhD type by DNA amplification. N Engl J Med 329507,199312. Arce MA, Thompson ES, Wagner S, Coyne KE, FerdmanBA, Lublin DM: Molecular cloning of RhD cDNA derived from agene present in RhD-positive, butnot RhD-negative individuals.

Blood 82:6Sl, 199313. Simsek S, Bleeker PMM, von dem Borne AEGKr: Prenataldetermination of fetal RhD type. N Engl J Med 330:795, 199414. Hyland CA, Wolter LC, Liew Y-W, Saul A: A Southernanalysis of Rh blood group genes: Association between restrictionfragment length polymorphism patterns andRh serotypes. Blood83566, 199415. Ciulla TA, Sklar RM, Hauser SL: A simple method for DNApurification from peripheral blood. Anal Biochem 174:485, 198816. Chtrif-Zahar B, Raynal V, Cartron JP, Colin Y: Organizationand expression of the Rh locus in Rh-deficient patients with Rh.,,,,D-- and DC- phenotypes. Br J Haematol 87:144, 1994 (suppl)

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