RHD Exon Research Articles

Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit

Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods--the NucliSens Magnetic Extraction (NMAG) system and the QIAamp DSP Virus Kit (QDSP)--against our current standard method, the widely used QIAamp DNA Blood Mini Kit (QDNA). The fetal DNA yield of the two extraction systems was evaluated using the RHD exon 7 as target in DNA extracts of 75 plasma samples from pregnant RhD-negative women, known to have given birth to RhD-positive infanto. The total DNA yield was evaluated in 23 samples, targeting GAPDH. The fetal DNA yield was improved by a mean factor of 1.7 using the NMAG system, and improved by a mean factor of 1.5 using the QDSP. The total DNA yield was improved by a mean factor of 2.3 using the NMAG system, and by a mean factor of 1.3 using the QDSP. Both extraction systems tested were superior to our standard with regard to DNA yield. This improvement may have a great impact on the success of genotyping in early pregnancy.

Molecular determination of RHD zygosity

Determining the RhD zygosity of a partner is valuable for the management of hemolytic disease of the newborn (HDN) related to anti-D. Zygosity is typically predicted using Rh phenotype. However, this may not be satisfactory in all ethnic groups. Our goal was to develop a robust clinical assay for RhD zygosity using molecular techniques that is suitable for use in both Caucasian and African-American populations. We used quantitative fluorescent PCR to detect RhD exons 5 and 7 with an internal control, exon 7 from the RhCE gene. PCR products were quantified by an automated sequencer. The ratio of exon 5 or 7 amplicon to the internal control determined RhD copy number. Accuracy was assessed using 17 Caucasian and 11 African-American samples whose zygosity was predicted from an Rh phenotype and 11 HDN paternal samples predicted heterozygous after the fetus tested RhD-negative. The assay accurately distinguished the 2 genotypes. Ratios for the DD and Dd genotypes for exon 5 and exon 7 were distinct and non-overlapping (Table). Common variant alleles such as DVI and the RhD pseudogene are recognized when the RhD copy number of exon 5 and exon 7 are discordant. All 17 Caucasian samples were concordant with serology. We found 2 discrepancies out of 11 African-American samples; however, the serology prediction for both cases was only 50-58%. All results were concordant for the 11 HDN paternal heterozygous samples. This molecular assay accurately assesses RhD zygosity and helps predict the risk that a fetus will inherit RhD.Tabled 1Exon Ratios Determine RhD Copy NumberExon 5Exon 5Exon 5Exon 7Exon 7Exon 7Copy#MeanRange3SDMeanRange3SDDD1.051.00-1.120.091.271.07-1.430.30Dd0.530.45-0.570.090.680.55-0.770.18 Open table in a new tab

Evaluation of prenatal RHD typing strategies on cell‐free fetal DNA from maternal plasma

The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women. The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity. Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.

In‐frame triplet deletions in RHD alter the D antigen phenotype

The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.

Fetal Blood Group Genotyping

Prediction of fetal blood group from DNA is usually performed when the mother has antibodies to RhD, to assess whether the fetus is at risk from hemolytic disease of the fetus and newborn (HDFN). Over the last five years RhD testing on fetal DNA in maternal plasma has been introduced. At the International Blood Group Reference Laboratory (IBGRL) we employ real-time quantitative polymerase chain reaction (RQ-PCR) to detect RHD exons 4, 5, and 10, which also reveals RHDpsi. SRY and, in RhD-negative (RhD-) females, eight biallelic polymorphisms are incorporated in an attempt to provide an internal positive control. Since 2000 we have tested 533 pregnancies for RhD. In 327 pregnancies where the RhD of the infant is known, we had one false-positive and one false-negative result. In 2004 we introduced fetal typing from DNA in maternal plasma for K, Rhc, and RhE, which represent single nucleotide polymorphisms (SNPs) on the KEL and RHCE genes. We have begun trials on an automated method for fetal RhD typing from DNA in maternal plasma. This is designed to test fetal RhD in all pregnant RhD- women, to identify the 40% with an RhD- fetus so that antenatal RhD immunoglobulin (Ig) prophylaxis can be avoided. Similar trials have already been reported by Sanquin Research Laboratories in Amsterdam.

The D category VI type 4 allele is prevalent in the Spanish population

The D category VI (DVI) is one of the clinically most important partial D. Three different molecular structures causing the DVI phenotype have been described. To determine the molecular basis of the DVI phenotype in the Spanish population, 20 DVI samples, previously detected in serologic screening, were examined by polymerase chain reaction with RHD exon-specific primers. Unexpected findings were further pursued by cDNA nucleotide sequencing. A novel pattern of RHD exon amplification was detected, which did not correspond to any of the previously described molecular structures. The cDNA sequence led to the identification of the new hybrid RHD-Ce(3-5)-D allele. The origin of exon 2 is undeterminable, because the 5' breakpoint was located within a region of RHD and RHCE identical sequence, which encompasses this exon. Sequencing of intron 5 allowed the 3' breakpoint to be mapped between the sixth and seventh polymorphic sites. Serologically, the hybrid protein has a D epitope expression pattern identical to the previously described DVI phenotypes and an antigen density slightly lower than DVI type 3. The new DVI variant is linked to the DCe haplotype and expresses the low-incidence BARC antigen. A novel structure causing the DVI phenotype, here named DVI type 4, has been characterized. This novel structure is the most frequent cause of DVI in Spain.

Detection of maternal deoxyribonucleic acid in peripheral blood of premature and mature newborn infants

Over the past decade, a lot of attention has been directed towards the fetomaternal and maternofetal transfer of nucleated cells and plasma DNA. In some autoimmune diseases, the fetal DNA is suspected to play an important role in the etiology of the disease. In the same way, the presence of maternal cells and free plasma DNA in fetal/newborn circulation gives rise to interesting questions. The aim of our study was to detect maternal deoxyribonucleic acid in the peripheral blood of premature and mature newborn infants. In the case of eight RhD-positive mothers-RhD-negative newborn pairs, peripheral blood samples were collected from the newborn infants within 35-120 min after birth. The maternal origin DNA was determined by real-time PCR amplification of the exon 7 of the RhD-positive allele. In all eight cases, the RhD exon 7 was amplified during the PCR reaction. The result of our study demonstrates that maternal DNA is present in the fetal peripheral circulation. The presence of maternally derived cells/DNA in the blood of newborn infants might have a role in the immunization of the newborn infants and also could be a possible explanation for 'grandmother effect' in the case of Rh-negative nulligravida patients.

Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative pregnant women

Prenatal noninvasive determination of fetal Rh status is an important aid to the management of hemolytic disease of the fetus and newborn. We performed real-time polymerase chain reaction on fetal DNA derived from maternal plasma to determine fetal Rh status. Cell-free plasma DNA from 98 D-negative pregnant women was tested for the presence of exons 4, 5, and 10 of RHD. The presence of fetal DNA was confirmed by detection of SRY or biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. Seventy-two D-positive infants and 26 D-negative infants were determined by serologic studies. All 3 RHD exon sequences were detected in 68 of 72 mothers of D-positive infants. The presence of fetal DNA in mothers of D-negative infants was confirmed in all 10 boys and in 14 of 16 girls. Fetal RHD genotyping in this study correctly predicted fetal Rh status in 92 of 98 (94%) cases.

Utilisation en routine clinique du génotypage fœtal RHD sur plasma maternel : bilan de deux ans d’activité

To evaluate the predictive value of RHD fetal genotype in maternal plasma of Rh D negative mothers after 10 weeks of gestation in a clinical use. Prospective, comparative study between fetal RHD genotyping in maternal plasma, with amplification of exons 4,5,10 of the RHD gene, by real-time multiplex PCR, and Rh D serology at birth, in 218 pregnancy and their 223 babies, between November 2002 and 2004. Combining the amplification of three exons, the concordance rate of fetal Rh D genotyping in maternal plasma and baby phenotyping at delivery was 100%. Four women whose the babies were Rh D negative were positive for RHD exon 10 during pregnancy. This positivity was, in three cases, correlated with the presence of RHDpsi pseudogene and in last case, with a haplotype Cdes (r's). RHD genotyping was performed for five twin pregnancies. Multiplex PCR using maternal plasma provides perfect prenatal prediction of fetal RHD gene. These results confirm that this non invasive procedure is the preferred method for assessing Rh D fetal status in Rh negative mothers. Using this method for two years in routine practice has led us to modify our management scheme for sensitized Rh D-negative pregnant women.

Random survey for RHD alleles among D+ European persons

RHD alleles are considered more variable in African persons than in European persons. A systematic survey, however, was lacking among D+ European persons at the molecular level, precluding any definite frequency estimate. A random survey was performed among 500 ccDee, 250 CcDee, and 250 ccDEe blood donors in southwestern Germany. They were tested by polymerase chain reaction with sequence-specific priming (PCR-SSP) for up to 12 single-nucleotide polymorphisms representative for the most frequent RHD alleles among European persons. The RHD exon 5 nucleotide sequence was also tested in all 1000 samples. The nucleotide sequence of the 10 RHD exons was checked in all samples with aberrant exon 5 or positive PCR-SSP procedures. By PCR-SSP, 15 aberrant RHD alleles were found among the 500 ccDee, 2 among the 250 CcDee, and none among the ccDEe samples. One of these was the novel RHD(F223V, E233Q, T379M) allele dubbed DAU-5. Weak D type 4 was detected more frequently than expected, whereas the population frequencies of the other RHD alleles conformed to published estimates. Nucleotide sequencing of RHD exon 5 further revealed three novel alleles RHD(G212G), RHD(R234W), and RHD(V245L), dubbed DUC-1, DQC, and DUC-2. In a limited screen at the molecular level among 1000 random D+ donors in southwestern Germany, 20 donors were found carrying aberrant RHD alleles. Four of these alleles were new and likely sporadic. An estimate was derived of the variety that may be encountered in genotyping approaches, and it was concluded that even within the European population the variety of RHD alleles may be larger than anticipated.

Aly/ REF, a factor for mRNA transport, activates RH gene promoter function

The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5' flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at -191 to -158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin-labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRalpha enhancer function. Aly/REF might be a novel transcription cofactor for erythroid-specific genes.

Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

Molecular characterization of D– Korean persons: development of a diagnostic strategy

D- frequencies show wide racial differences: in particular, in Korean persons it is approximately 1/100th of that in Caucasians (0.15% vs. 15%). The molecular mechanisms of D- may be unique to races, and thus specific molecular diagnostic approaches are necessary for individual races. Such marked racial differences have been attributed to the founder effect. It was hypothesized that D- frequencies may be affected by genomic variations of Rhesus boxes, which are instruments of unequal crossing-over. First, the molecular basis of D- in Korean donors was characterized. A total of 264 D- persons were analyzed by performing RHD exon polymerase chain reaction (PCR), Rhesus box hybrid PCR-PstI, RHD sequencing, and a novel RHD-CE-D hybrid PCR. Second, with sequencing analysis, Rhesus boxes in Korean and Caucasian persons were compared. Of 264 D- Korean individuals, 74 percent completely lacked RHD, 9 percent had RHD-CE-D hybrid, and 17 percent had point mutations. Three genetic causes, RHD deletion, RHD-CE(2-9)-D2, and 1227G>A, explained 99.4 percent of D- alleles in Korean persons. Three novel mutations were also found. The identity region of 10 Rhesus boxes in Korean persons was in complete concordance with that in Caucasian persons. In this study, a molecular diagnostic strategy was established and the genetic causes of almost all D- Korean persons was able to be diagnosed with a simple decision tree. The study provides a good example of the molecular diagnosis of D- persons, especially in low-frequency areas. Our hypothesis that D- frequencies are affected by genomic variations in Rhesus boxes was excluded. Rather, racial differences may be influenced by the founder effect.

Systemic analysis and zygosity determination of the RHD gene in a D‐negative Chinese Han population reveals a novel D‐negative RHD gene

The aim of this study was to systemically analyse the genetic background of D negativity in a Chinese Han population. DNA of 74 D-negative samples was analysed by using an RHD multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by PCR-restriction fragment length polymorphism (PCR-RFLP) for RHD zygosity determination. Sixty-five samples were additionally analysed by using real-time quantitative PCR on RHD exon 7. RHD exon-specific sequencing was performed on discrepant samples. Forty-six samples (62%) showed the absence of RHD-specific exons by RHD MPX PCR and homozygous RHD negativity by PCR-RFLP. Twenty-two samples (30%) showed a 1227G>A mutation, characteristic for the Del phenotype. Five (7%) samples showed all characteristics of the RHD(1-2)-CE(3-9)-D(10) hybrid gene. One sample (1.4%) showed a novel 933C>A nonsense mutation in RHD exon 6, which resulted in a premature stop codon. The RHD gene deletion, RHD-CE-D hybrid genes and one novel 93C>A mutation were found to be the three mechanisms that cause D negativity in our samples. The 1227G>A Del mutation was found to be the major cause of discrepant results between genotyping and phenotyping strategies, favouring genotyping of D-negative samples.

Reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women

The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis. A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th-36th week of gestation. Thirty-eight samples were from ongoing pregnancies of Danish women and 21 samples from 18 pregnant women were stored anonymized samples from the International Blood Group Reference Laboratory, Bristol, United Kingdom. Prediction of fetal RhD type was compared with the serological result obtained after birth. The prediction of the fetal RhD type was in 100% concordance with the serological RhD type from the 16th week of gestation. One sample from the 15th week of gestation was inconclusive. The number of copies of fetal RHD DNA was found to increase with gestational age. Low levels of DNA were found to follow the Poisson distribution (p = 1.0000). Our set-up was very reliable for determination of fetal RhD genotype, and thus will be of value in prenatal Rh prophylaxis and in the management of immunized women.

RHD Genotyping of Blood Donors May Avoid Anti-D Immunization.

Antigen D compatible transfusion is standard practice in modern transfusion therapy, warranting proper antigen D typing in all blood donors. Blood group genotyping is increasingly utilized for prenatal diagnosis and after recent transfusions. RHD genotyping is of practical importance to overcome the limitations of standard serology, which frequently fails to detect some weak D and chimeric red blood cell (RBC) populations, and to enhance clinical safety for blood transfusion recipients. Recently, the transfusions of blood units from chimeric donors were reported to having induced an acute transfusion reaction and multiple anti-D immunizations. The latter donor escaped the serologic detection in 13 donations but was uncovered by the strategy reported in this study. Since January 2002 all D negative (D neg.) first time donors at blood center A were tested for carrying the RHD gene. We evaluated the results of this two years' routine testing and compared them to a screening study conducted in the independent blood center B. This approach contributed to define the utility of RHD genotyping for the quality control of D neg. RBC units.In two independent blood centers we examined 9,931 and 5,115 serologic D neg. blood donors. Samples were tested in pools of 20 donors by PCR-SSP for RHD intron 4 or for RHD exon 4, exon 7 and exon 10. 21 RHD positive donors were detected in center A and 18 RHD positive donors in center B among the serologic D neg. donors. The molecular bases of the RHD positive samples were resolved in all cases. A total of 10 RHD alleles were novel: RHD(T201R, F223V, P291R) dubbed weak D type 4.3, RHD(I374N) dubbed weak D type 32, RHD(del147), RHD(del343), RHD(del449), RHD(del785), RHD(L153P), RHD(Y269X), RHD(IVS3+2T>A) and RHcE(1–3)-D(4–10). 13 samples in center A represented 5 known RHD alleles, most often RHDψ (n = 5), RHD(IVS3+1G>A) (n = 4) and RHD(M295I) in CDe (n = 2); 7 samples in center B represented the 3 known RHD alleles RHD(IVS3+1G>A) (n = 4), RHD-CE(2–9)-D2 (n = 2) and RHD(M295I) (n = 1). 9 donors in center A represented Del; in center B 9 were weak D and 6 Del; 13 of the remaining donors were confirmed to be D neg. despite carrying the RHD gene. We concluded that RHD genotyping of serologic D neg. donors at two facilities revealed carriers of the RHD gene expressing antigen D, albeit at low levels, in the range of up to 1:1,000 and 1:350 donors, respectively. At least 24 donors carried RHD alleles that were known or shown to express a weak D or Del phenotype. The RBC units donated by these donors may be capable of causing at least secondary anti-D immunization. This possible adverse clinical outcome was avoided by RHD genotyping each donor only once. Use of RHD genotyping would obviate the need to tightly control the sensitivity of serologic anti-D testing in blood donors. Further studies are needed to corroborate the current experience in particular in donors of non-white ethnic background. However, we think that RHD genotyping in first time blood donors has the potential to become a routine procedure in blood centers.

Molecular basis of RhD-positive/D-negative chimerism in two patients

This study investigated two patients with Rh chimerism: patient A, a healthy individual, and patient B with myelofibrosis. Flow cytometry studies showed two red blood cell populations of Rh phenotypes R1r and rr at percentages of about 25% and 75% respectively. Normal RhD transcript sequences were found following RT-PCR. Genomic DNA (gDNA) showed normal exon, intron, GATA regions and exon/intron boundary sequences except for a single base change in intron 7 (C --> A) of exon 7 in patient A. The major change found in both patients was the absence of RHD exon 9 DNA in gDNA isolated from peripheral blood. These findings suggest a somatic mutation, probably in a stem cell common to the myeloid lineage of both patients, and indicate that patient A may undergo malignant transformation in the future.

Molecular basis of weak D in Taiwanese.

Two genes, RHD and RHCE, encode the antigens of the RH blood group system. The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, resulting in distinct serologic phenotypes and anti-D immunization. We analyzed seven weak D phenotypes excluding D(el), using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods to detect the changes of all ten RHD exons. The results show that there are four types of weak D in Taiwanese: one case each for CGG to CAG mutation at codon 10, GTG to ATG mutation at codon 174, and GTG to GAG mutation at codon 270, and four cases for GGT to GAT mutation at codon 282. In conclusion, we present the first data of a molecular basis of weak D in Taiwanese, which suggest a clinically relevant potential for anti-D immunization and may improve transfusion strategy in weak D Taiwanese patients.

RHCE-D-CE hybrid genes can cause false-negative DNA typing of the Rh e antigen.

DNA typing of the human Rh blood groups generally shows good agreement with serologically defined phenotypes. However, in the present report we describe four individuals who were declared Rh e negative by genotyping although they express the Rh e antigen. Serotyping was performed using mono- and polyclonal Rh antisera. Fluorescent multiplex sequence-specific polymerase chain reactions (PCR-SSPs) identified RHD exons and the polymorphisms usually associated with the Rh E/e or Rh C/c/C(W) antigens. Additional PCR amplification reactions, which were carried out to reveal RHCE-D-CE hybrid genes, analysed exon 5 of the RH genes, the location of the polymorphism (676C-->G) coding for the Rh E and Rh e antigens. Four individuals were identified who expressed Rh e antigens but were negative by PCR-SSP typing for common Rhe-coding sequences. In one family analysed in detail, an RHCE-D5-CE hybrid gene associated with Rh e antigen expression was identified. A concomitant RHcE allele accounted for a seemingly regular typing pattern by conventional RH PCR. The presence of RHCE-D5-CE hybrid alleles may cause false-negative DNA-typing results for the Rh e antigen that are easily overlooked unless appropriate RH hybrid PCR-SSPs are incorporated into conventional DNA-typing protocols. These and previous data strongly caution against an uncritical interpretation of RH DNA-typing results.

Early detection of RhD status in pregnancies at risk of hemolytic disease of the newborn.

The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anti-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and I DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided.