The Genomic Organization of the Partial D Category DVa: The Presence of a New Partial D Associated with the DVaPhenotype

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Biochemical and Biophysical Research Communications 254, 786–794 (1999)

Article ID bbrc.1998.0121, available online at http://www.idealibrary.com on

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he Genomic Organization of the Partial D Category D :he Presence of a New Partial D Associatedith the DVa Phenotype

oshinori Omi,*,1 Junko Takahashi,† Naoki Tsudo,* Hiroshi Okuda,* Sadahiko Iwamoto,*itunobu Tanaka,† Taiko Seno,‡ Yoshihiko Tani,† and Eiji Kajii*

Department of Legal Medicine and Human Genetics, Jichi Medical School, Minamikawachi-machi, Tochigi,29-0498, Japan; †Osaka Red Cross Blood Center, Morinomiya, Joto-ku, Osaka, 536-8505, Japan; andReference Murakami Laboratory, Toyo, Koto-ku, Tokyo, 135-0016, Japan

eceived December 25, 1998

Within the Rh blood group, the partial D phenotypeiiiDRcaamrObDTtD

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Within the Rh blood group, the partial D phenotype iswell known RhD variant, that induces Rh-incompatiblelood transfusion and hemolytic diseases in the new-orn. The partial D category DVa phenotype (DVa Kou.)esults from a hybrid of RhD-CE-D transcript. We dem-nstrated a genomic organization of the hybrid RHD-E-D gene leading to the DVa phenotype, and showed

hat the DVa gene were generated from gene conversionetween the RHD and the RHCE genes in relativelymall regions. This study also revealed that the presencef a new partial D associated with the DVa phenotype,hich we termed the DVa-like phenotype. In this pheno-

ype, five RHD-specific nucleotides were replaced withhe corresponding RHCE-derived nucleotides on thexon 5 of the RHD gene. In addition, two variants of theutated RHD genes at nucleotide 697 were revealed in

he RhD variant samples. These results will provide use-ul information for future research into the diversifica-ion of the Rh polypeptides. © 1999 Academic Press

The human Rh blood group system is a clinicallymportant and polymorphic blood group (1,2). This sys-em consists of two highly homologous genes (RHD andHCE) encoding 30 kDa polypeptide (3–5). Both theHD and the RHCE genes are composed of 10 exons

6), and locate at chromosome 1p34-1p36 (7,8). ThehD mRNA encodes the RhD antigen, while the RhCERNA encodes both the RhC/c and RhE/e antigens

9–15). The RhD and RhCE peptides differ by 35 sub-titutions of 417 amino acids and exhibit a similarembrane topology with twelve putative transmem-

rane helixes. The RHD gene is completely deleted inhe RhD negative-Caucasian donors (16) but found inome Australian, Japanese, and African RhD negativeonors (17–19).

1 To whom correspondence should be addressed. Fax: 181-285-44-902. E-mail: t-omi@jichi. ac.jp.

786006-291X/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

s a well known RhD variant, that and induces Rh-ncompatible blood transfusion and hemolytic diseasesn the newborn (20,21). In serologic testing, the partial

erythrocytes are reactive against polyclonal anti-hD antibodies, but inactive with some of the mono-lonal anti-RhD antibodies. The partial D phenotypesre classified into different categories according to thebsence of one or more RhD epitopes (22–24). Recentolecular analysis of the partial D transcripts has

evealed two genetic mechanisms leading to partial D.ne genetic mechanism is the gene conversion eventsetween the RHD and the RHCE genes: DVI (25–29);IIIb (30); DIVa, DIVb, DVa, DFR (31); DIIIc (32); DBT (32).he other genetic mechanism is the point mutations inhe RHD gene; DVII (34,35); DHMi (36); DII , DNU (37);

IIIa (38); DHR (39).Rouillac et al. reported that the partial D category DVa

henotype appeared to result from a gene conversionvent between the RHD and RHCE genes in two DVa

aucasian donors (31). The hybrid RhD-CE-D tran-cripts in which the complete exon 5 of the RHD geneRHD exon 5) or a portion of it has been replaced by theorresponding region of exon 5 of the RHCE gene (RHCExon 5). However, the hybrid site of the RHD-CE-D genen DVa phenotype has not been defined yet. In this paper,e performed a genomic organization of the RHD gene

rom DVa donors, and showed that the DVa gene wereenerated from gene conversion between the RHD andhe RHCE genes in relatively small regions. In addition,e descovered a new partial D associated with DVa phe-otype, which we termed the DVa-like phenotype.

ATERIALS AND METHODS

Blood sample. Agglutination studies were performed using variousonoclonal antibodies (MoAbs) in Osaka Red Cross Blood Center

Osaka, Japan). The monoclonal antibodies (MoAbs) OSK3, OSK3-1,SK3-2 (Osaka Red Cross Blood Center), and HIRO-1, HIRO-2,

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Vol. 254, No. 3, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IRO-3, HIRO-4, HIRO-5, HIRO-6 (Central Red Cross Blood Center,okyo, Japan), which were used for analysis (see Table 1). The RBCsere positive with polyclonal anti-D (Ortho Diagnostic Systems, NJ,SA). The RBCs from A. K. and Y. H. were obtained from Osaka Redross Blood Center. Sample K. R. was found to be weakly reactive witholyclonal anti-D but was not tested using monoclonal antibodies. TheBCs from N. N. and H. K. were obtained from Toyama Red Crosslood Center (Toyama, Japan). The RBCs from H. B., H. O., M. K.,. S., and S. M. individuals were obtained Nagasaki Red Cross Bloodenter (Nagasaki, Japan), Himegi Red Cross Blood Center (Hyogo,apan), Miyazaki Red Cross Blood Center (Miyazaki, Japan), Saitamaed Cross Blood Center (Saitama, Japan) and Aichi Red Cross Bloodenter (Aichi, Japan), respectively.

Polymerase chain reaction (PCR) and reverse-transcription PCR.enomic DNA was isolated from donor peripheral leukocytes (40).he RHD exon 5 was amplified by nested-PCR. The first PCR waserformed in total volume of 50 ml containing 200 mM of dNTPs, 0.24M of primes (4Ds/PI5-95a), 5U Taq DNA polymerase (Toyobo, To-yo, Japan), and 5 ml of 10 X Taq buffer (15 mM MgCl2, 100mMris-HCl pH 8.3, and 500 mM KCl). The PCR amplification waserformed under the following conditions; 1 minute at 94°C, 1inute at 55°C, 1 minute at 72°C for 35 cycles. The second PCR was

erformed in total volume of 50 ml containing 5 ml of first PCRroduct, 200 mM of dNTPs, 0.24 mM of primes (PI4-112s/PI5-41a), 5Uaq DNA polymerase, and 5 ml of 10 X Taq buffer. The PCR conditionf the second PCR was same as that of the first PCR. The intron 4 ofhe RHD gene (DNA segment encompassing exons 4 through exon 5)as amplified by the nested PCR using two primer sets of P4Ds/PI5-5a, and P4s/PI5-41a on the standard PCR conditions.Total RNAs were isolated from DVa RBCs (A.K.) using the acid-

henol-guanidium method (41). Reverse transcription-PCR (RT-CR) was performed using a one step RT-PCR kit (Takara, Shuzo Co.TD, Shiga, Japan) according to the manufacture’s instructions.NAs were reverse-transcribed for 60 minutes at 50°C in total vol-me of 25 ml of a reaction mixture containing 2.5 U of the Avianyeloblastosis Virus (AMV) reverse trancriptase, 20 U of RNase

nhibitor, 10 X RNA PCR, 2.5 U of AMV-Optimized Taq, 2.5 ml of 10RNA PCR buffer, 5 ml of 25mM MgCl2, 2.5 ml of 10mM dNTPs, and

.24 mM of primes (P1-1s/P39UTD). The primer sequence of P39UTDas described in previous report (42). RT-PCR amplification was

787

erformed under the following conditions; 2 minutes at 94°C for 1ycle, 1 minute at 94°C, 1.5 minutes at 55°C, 3 minutes at 72°C for5 cycles, and 10 minutes at 72°C for 1 cycle. After the RT-PCR, thehD cDNA was amplified the PCR using primer sets of P1s/P6Da or4Ds/P1254a. Total volume of 50 ml PCR mixture containing 5 ml ofT-PCR product, 200 mM of dNTPs, 0.24 mM of primes, 5U Taq DNAolymerase, and 5 ml of 10 X Taq buffer. PCR amplification waserformed under the following conditions; 1 minute at 94°C, 1.5inutes at 55°C, 3 minutes at 72°C for 35 cycles, and 10 minutes at

2°C for 1 cycle. The PCR amplified fragments were analyzed on 2%garose gels, stained with ethidium bromide.

Subcloning and DNA sequence analysis. The PCR amplified frag-ents were purified using 1.5% low-temperature melting gel, and were

ubcloned into pCR II (Invitrogen, San Diego, CA) using the supplier’snstructions. After transformation, the clones with insert were screenedy color selection. Plasmid DNAs with positive insert were isolated byhe alkaline lysis method, and six or more independent clones of eachubcloned fragment were sequenced using a DNA sequencing kit (USB,leveland, Ohio) according to the dideox termination method (43). A 6%olyacrylamide gel containing 8 M urea was used for electrophoresis.

Allele-specific primer amplifications (ASPA). The four sets ofllele-specific primer were designed for RHD typing on exon 5. PCR-SPA using primer sets of PI4-112s/697RD, PI4-112s/697RC1, andI4-112s/697RC2 were performed under the following conditions; 5inute at 94°C for 1 cycle, 1 minute at 94°C, 45 seconds at 67°C, 30

econds at 72°C for 30 cycles, and 5 minutes at 72°C for 1 cycle.CR-ASPA using a primer set of PI4-112s/697RN was performed underhe following conditions; 5 minute at 94°C for 1 cycle, 1 minute at 94°C,0 seconds at 67°C, 30 seconds at 72°C for 30 cycles, and 5 minutes at2°C for 1 cycle. In addition, we also performed PCR-ASPA using sixets of allele-specific primer to amplified exons, 3, 4, 6, 7, 9, and 10 forHD typing. Each primer sets were P3Ds/PD3a, P4Ds/P4Da, P6s/6Da, P7Ds/P7Da, P9Ds/P9Da, and P10Ds/P10Da. Exon typings of 3, 7,nd 10 were performed according to our previous method (18). Exonypings of 4, 6, and 9 were carried out under the following conditions; 5inute at 94°C for 1 cycle, 30 seconds at 95°C, 30 seconds at 67°C, 30

econds at 72°C for 35 cycles, and 10 minutes at 72°C for 1 cycle.CR-ASPA were performed in total volume of 50 ml containing 200 mMf dNTPs, 0.24 mM of primes, 5U Taq DNA polymerase (Toyobo, Tokyo,

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apan), and 5 ml of 10 x Taq buffer (15 mM MgCl2, 100mM Tris-HCl pH.3, and 500 mM KCl). The PCR amplified fragments were analyzed on% agarose gels, stained with ethidium bromide. Sequences and loca-ions of primers are shown in Table 2.

ESULTS

Serology. Nine RBCs including the DVa phenotypeere investigated serologically with some panel ofonoclonal anti-D according to presence or absence of

pitopes. The results of the serological test of the RBCsrom H. O. corresponded to of the DVa RBCs from A. K.nd H. B. The RBCs from A. K. and H. B. have beenharacterized as DVa phenotype in Medical Researchouncil Blood Group Unit (MRC, London, U.K.). TheBCs from M. K., Y. H., K. S., and N. N. were catego-

ized into the same group, and were similar to all DVa

BCs except for aside from anti HIRO-6 by the agglu-ination test. These results suggested that these RBCsere a new partial D associated with DVa phenotype inepiotpe model. This phenotype was termed the DVa-

ike phenotype by the authors of the present study. Thegglutination pattern of the RBCs from S. M. and H. K.howed an absence of some RhD epitopes but could note identified within the partial D category. These se-ological data are shown in Table 1. The Rh phenotypesf A. K., H. B., H. O., Y. H., K. S., M. K., N. N., S. M.,

788

nd H. K. individuals were Ccee, CcEe, Ccee, CCee,CEe, Ccee, CcEe, Ccee, and Ccee, respectively. Ourerological investigation suggested that the RBCs fromach donor caused the alternation of the Rh peptide onhe 4th loop, we analyzed the RHD gene on exon 5 fromhe genomic DNA of each donor.

Isolation and sequencing for exon 5 of the RHD gene.xon 5 of the RHD gene was amplified using theested-PCR method using the primer sets of P4Ds/PI5-5a and PI4-112s/PI5-41a (Fig. 1), and PCR fragmentsf 515 bp were detected in all samples (Fig. 2). In the

Va RBCs of A. K., H. B., and H. O. individuals twoHD-specific nucleotides-667 and 697-were replacedy the corresponding RHCE-derived nucleotide, whichorresponded to the type of DVa kou (31). In contrast,he RHD-specific nucleotides-667, 697, 712, 733, and44-were replaced by the corresponding RHCE-deriveducleotide in the DVa-like RBCs from M. K., Y. H., K. S.,nd N. N., and a K. R. sample. In addition, we revealedne point mutation of G to C and G to A at nt 697 of theHD gene in the S. M. and H. K., respectively. The97C was an RHD specific nucleotide but the 697A didot corresponded to either RHD nor RHCE specificucleotide. The results of these sequence analysis ofxon 5 of the RHD gene are shown in Fig. 3.

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Isolation and sequence analysis of intron 4 of theHD gene in DVa and DVa-like phenotypes. We showed

hat a part of RHD exon 5 was replaced by the RHCExon 5 in DVa and DVa-like phenotypes, and that theybrid site of the RHCE-D were located in exon 5. To

nvestigate the hybrid site of the RHD-CE, the frag-ents spanning from exons 4 and 5 were amplified

rom genomic DNAs by nested-PCR. After the PCRragment of the 748-bp products were detected, theCR product were subcloned and sequenced. In thisegion, the nucleotides at nt 143, nt 208, and nt 246ere a, g and t, respectively. The RHCE extra sequence

ncluding the Alu repeat were deleted. These se-uences were identical with that of the intron 4 of theHD gene. The 707 nucleotide sequences encompass-

ng exons 4 and 5 including intron 4 are shown in Fig.. A schematic representation of the gene conversionvents between the RHD and RHCE genes in the DVa

nd DVa-like phenotypes is shown in Fig. 5. The geneonversion between the RHD and the RHCE genes

FIG. 1. Schematic representation of the strategy for the genomichenotypes. Boxes and numbers show the exons of the RHD gene. Arimers are shown in Table 2.

FIG. 2. The PCR amplification for exon 5 of the RHD gene fromxon 5, the 515-bp band were revealed in all samples. The PCR prodels, and stained with ethidium bromide. The size marker (M) is thef D positive- and D negative-blood donors were shown by RhD (1)

789

rised in relatively small regions. In S. M. and H. K.,he intron 4 sequence also corresponded to that of RHDntron 4 (data not shown).

Predicted amino acid sequences. The amino acidaa) substitutions, as compared to the normal RhDolypeptide, were indicated at the following positionsn each donor. The DVa donors exhibited two amino acidubstitutions in the RhD polypeptide: Phe2233Val223,lu2333Gln233. The DVa-like donors exhibited fourmino acid substitutions in the RhD polypeptide:he2233Val223, Glu2333Gln233, Val2383Met238, andal2453Leu245. The S. M. and H. K. individuals showedne amino acid substitution in the RhD polypeptide:lu2333Gln233, and Glu2333Lys233. The predicted topol-gies of the DVa and DVa-like phenotypes are shown inig. 6. Full length RhD-cDNA from A.K. RBCs with theVa phenotype was sequenced and represented by alack line. The sequence data from the cDNA wasorresponded with that from genomic DNA.

anization of the RHD gene with partial D category DVa and DVa-likeA is allele specific primer amplification. Sequences and positions of

RhD variants samples including DVa donors. After amplification ofts amplified from the genomic DNAs were separated on 2% agarose

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Allele-specific primer amplifications (ASPA). A se-uence analysis of the RHD exon 5 revealed that theucleotide at 697G was changed to C or A in each donorFig. 3). To detect for this mutation by PCR genotyping,e performed allele-specific primer amplifications

ASPA) using four primer sets. Fig. 7A showed that thepecific fragment for the RHD exon 5. The PCR productas amplified from the Rh D positive donors. The ex-ected size of PCR product was 397bp. In the DVa

onor, the DVa-like donor, and S. M. individual, thepecific fragment for the RHD exon 5 was not ampli-ed, whereas the hybrid fragments of the RHD-CEene were revealed using the primer set of PI4-112s/97RC (Fig. 7B). In the H. K. individual, the PCRroduct was amplified using the primer set of PI4-112s/97RN (Fig. 7C). Figure 7D shows the result of ampli-cation for each of the exons of the RHD gene in the. K. RBCs with the DVa phenotype. The fragments

rom exons 3, 4, 6, 7, 9, and 10 were detected as normalands. The size of each product was 134bp, 128bp,33bp, 95bp, 73bp, and 198bp, respectively. The re-ults of PCR-ASPA for DVa and DVa-like donors weredentical with those of the A. K. RBCs with the DVa

henotype.

FIG. 3. Schematic representation of the nucleotide sequences oevealed in the DVa phenotype of A. K. and H. B., and the H. O. indivetected in the DVa-like phenotype of M. K., Y. H., K. S., and N. N.,t al (31). One point mutations of G to C and G to A at nt 697 were dre shown by (*). Nucleotide sequences of A. K. (DVa), K. R. and Y. Humbers are AB012623, AB012658, AB012769, and AB012659, resp

FIG. 4. The nucleotide sequences of the encompassing exons 4 anenomic DNAs of the DVa phenotypes derived from samples A. K., aifferences between RHD and RHCE genes are indicated by boxes.HCE genes. The nucleotide of the RHD and RHe genes is same at oucleotide 295 through 943 in the intron 4 was the RHCE specific reere submitted to Gene Bank; accession numbers are AB012661 (A

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ISCUSSION

Among the partial D categories, the DVa phenotype isnown to be defective in epitopes 1 and 5 (under the-epitope model) or epitopes 1, 2, 7, 8, 9, 10, 11, 26, 31,nd 32 (under the 37 epitope model) and to be detective ofhe Rh23 (Dw) antigen by serological reactivity usingonoclonal antibodies (44–48). From a molecular basis

or the partial D category DVa phenotype, the hybridhD-CE-D transcripts in which the complete RHD exonor a portion of it has been replaced by the corresponding

egion of the RHCE exon 5. The genetic mechanism foreading to the partial D categories are explained by theene conversion events between the RHD and RHCEenes or the point mutations in the RHD gene. In theresent study, we investigated the hybrid site of theHD-CE-D gene leading to Dva phenoitype with with DVa

Kou). The 573G was derived from the RHCE gene buthe upstream region from the nucleotide at 295 was notn RHCE specific sequence. The gene conversion theHD gene to RHCE gene occured in a region encompass-

ng nucleotide 295 through 572. In contrast, the 603Cas an RHCE derived nucleotide whereas the 618A wasn RHD derived nucleotide in the DVa gene. These data

on 5 of theRHD gene. Two mutations at nt 667 and nt 697 wereals. Five mutation at nt 667, nt 697, nt 712, nt 733, and nt 744 were

a sample K. R. individuals. # DVa (Kou.) was reported by Rouillaccted in samples of S. M. and H. K. Mutated nucleotides in all typesDVa-like), S. M., and H. K. were submitted to Gene Bank; accessionively.

xon 5 of the RH gene encoding the DVa and DVa-like phenotypes. Thethat of DVa-like phenotype did from K. R. and Y. H. The nucleotideen triangle is showed the nucleotide difference between RHD andtriangle. The nucleotide deletion are indicated by (.). The spanning

n, which was deleted in the RHD gene. These nucleotide sequences) and AB012660 (K. R. and Y. H.).

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emonstrated that the DVa gene was on the RHD-CE-Dybrid gene that results from a in the segmental DNAxchange through gene conversion events between theHD and RHCE genes in a relatively small region

Fig. 5).We also demonstrated the presence of a new partialassociated with DVa phenotype. In the 9 epitope

odel, the agglutination pattern of the RBCs from fourndividuals were very similar to that of the DVa RBCssing our monoclonal anti-D aside from the HIRO-6ntibody. Thus the new partial D phenotype wasamed the DVa-like phenotype by the authors of theresent study. A serological difference between the DVa

nd DVa-like phenotypes could also be confirmed theequence analysis. The amino acid substitutions at 223nd 233 were exhibited both the DVa and DVa-like phe-otypes, whereas the amino acid substitutions at 238

FIG. 5. Schematic representation of the gene conversion events bhe black box showed the fragments spanning from intron 4 and exonresented in Fig. 4. The hatched box indicated the RHCE specific served as the donor and the RHD as the recipient gene.

FIG. 6. Proposed topology of amino acid substitutions on the 4thoop of the RhD polypeptide in the DVa and DVa-like phenotypes. Theapanese DVa donors were corresponded to DVa (Kou.) (31). The blackine showed the sequencing detected by cDNA analysis. Black circles the RhD to the RhCE substitutions.

792

nd 245 were only reveled in the DVa-like phenotype. Aopology investigation of the Rh polypeptide revealedhat the aa 238 was located in the exofacial domainhile the aa 245 was located in the intramenbraneomain (49). These results suggest that the antiIRO-6 recognized Methionie at 238 located in the

xofacial domain. In the Dva phenotype, two variants ofhe DVa gene were revealed in DVa (Hus) and DVa (Kou).

complete RHD exon 5 including seven RHD-specificucleotides are replaced by the corresponding RHCE-erived nucleotide in DVa (Hus). In contrast, a portionf the RHD exon 5 including two RHD-specific nucleo-ides are replaced by the corresponding RHCE deriveducleotide in DVa (Kou). The DVa-like phenotype, whichesults from five nucleotide substitutions in the RHDxon 5, showed the middle type of the DVa (Hus) and

Va (Kou). Thus the DVa-like phenotype may be thehird of the DVa phenotypes. For further analysis, theetection of the Dw antigen (Rh23) on the membrane ofhe DVa-like RBCs is needed.

The serological characteristics of the RBCs from the. M. and H. K. individuals differed from those of bothhe DVa and DVa-like RBCs. In our serological study, weere not able to establish any partial D category withnucleotide sequence of the RHD gene of exon 5 that

id not correspond to any reported previously partial Dategory. One amino acid substitution was detected ata 233 on the 4th loop of the Rh polypeptide in the. M. and H. K. individuals. As this amino acid substi-ution was also detected in DVa and DVa-like pheno-ypes, these variant samples will be usuful tools foresearch into the expression analysis of Rh epitopes.Gene conversion events seen to have the further poten-

ial to generate either templated- or untemplated-singleucleotide replacements to generate additional diversifi-

een the RHD and RHCE genes in the DVa and DVa-like phenotypes.f the RH genes, which was compatible with the nucleotide sequencesences in intron 4. In the case of the hybrid RHD-CE-D, the RHCE

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ation (50). Previous reports have shown untemplatedutation occurred as a result of incorrect repair of aetelroduplex formed between the recombining se-uences, and such a mechanism was proposed for therigin of some glycophorin variants such as MiI or MiIX,r as a mechanism for diversification of major histocom-atibility or immunoglobulin gene families (51–54) Anmino acid substitution at 233 is caused by a singleucleotide replacement at 697 of the RHD gene in the. M. and H. K. individuals. The point mutation of the

FIG. 7. Allele-specific primer amplifications (ASPA) of the RHDene. (A) ASPA for RHD exon 5 (PI4-112s/697RD). In all types, the97bp product did not detected. (B) Amplification of the RHD-CE hybridene on exon 5. (PI4-112s/697RC). The 397-bp products from RHD-CEene were revealed in the DVa donors, DVa-like donors, and the S. M.ndividual. (C) Amplification of the RHD mutant gene carried withdenine at nucleotide 697 (PI4-112s/697RN). The 397-bp product wereevealed in the H. K. individual. (D) RHD typing on exons 3 (P3Ds/3Da), 4 (P4Ds/P4Da), 5 (PI4-112s/697RD), 6 (P6s/P6Da), 7 (P7Ds/7Da), 9 (P9Ds/P9Da), and 10 (P10Ds/P10Da). PCR products except forxon 5 were amplified from exons 3, 4 , 6, 7, 9, and 10 in all samples. Theize of each products was 134bp, 128bp, 133bp, 95bp, 73bp, and 198bp,espectively. The result of PCR was showed the A. K. individual withhe DVa phenotype. The PCR products amplified from the genomicNAs were separated on 2% or 3% agarose gels, and stained withthidium bromide. The size marker (M) is the Hae III-cleaved frag-ents of 174 replicative form DNA.

793

hat of H. K. did not corresponded to RHCE- nor RHD-erived residue. Thus these variants could have arisen bytemplated or an untemplated nucleotide replacement

esulting from a gene conversion event, respectively. Theene conversion events resulted in segmental replace-ents occurred at a length of between one (minimum) to

4 nucleotides (maximum). Moreover, the gene conver-ion accompanied by an untemplated mutation is be-ieved to be a possible mechanism for the altered residuet aa 233 in the H. K. individual.Various Rh D genotyping techniques were applied to

enomic DNA samples derived from the expression ofifferent Rh phenotypes; D1, D2, partial D, and weakphenotypes (55–60). In general, this was done sincesingle RHD typing could potentially generate a false-ositive or false-negative result. Thus, RHD genotyp-ng assay must be done in at least two or more regionsf the RHD genes. The present study also detected thepresence of a nucleotide mutation at nt 697 in the DVa,

Va-like, and other RhD variant samples. The allele-pecific primer amplification targeted at nt 697 shoulde observable by RHD genotyping assay done to detecthe DVa phenotype or the mutation of the RHD gene.he development of the DVa genotyping should providesuful for research into the clinical significance of theVa phenotype (61).

CKNOWLEDGMENTS

We thank the stuff of Osaka Red Cross Blood Center (Osaka,apan), Aichi Red Cross Blood Center (Aichi, Japan), and Toyamaed Cross Blood Center (Toyama, Japan), Saitama Red Cross Bloodenter(Saitama, Japan), Miyazaki Red Cross Blood Center

Miyazaki, Japan), Nagasaki Red Cross Blood Center (Nagasaki,apan) for serological testing or for gift of DVa and DVa-like samples.e thank Dr. Patricia Tippett, Medical Research Council Bloodroup Unit (MRC, London, U. K.) for identification of the DVa phe-otype in the A. K. and H. B. samples. Especially, MT. Tadao Tomita,T. Hajime Yamano, MT. and Toru Nakade (Osaka Red Cross Bloodenter) for serological testing of DVa and DVa-like samples and Dr.akoto Uchikawa (Central Red Cross Blood Center, Tokyo, Japan)

or gift of the monoclonal antibodies. We also thank Takashi Oya-ada for excellent technical assistance (Jichi Medical School,ochigi, Japan). This work was supported by a Grant-in Aid (No.9557041, No09671129) for scientific research from the ministry ofducation, science and culture of Japan.

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Documents

The Genomic Organization of the Partial D Category DVa: The Presence of a New Partial D Associated with the DVaPhenotype