Prenatal Determination of Fetal RhD Type by DNA Amplification

Bennett, Phillip R.; Kim, Caroline Le Van; Colin, Yves; Warwick, Ruth M.; Chérif-Zahar, Baya; Fisk, Nicholas M.; Cartron, Jean‐Pierre

doi:10.1056/nejm199308263290903

Cited by 254 publications

(103 citation statements)

References 24 publications

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“…Four PCR methods, I, II, III and IV, were performed to evaluate genomic RHD and RHCE [11, 17, 20]. The four methods amplified exons 4, 5, 7 and 10.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR method I was performed as described by Bennett et al [20]with a slight modification of the PCR conditions. The following primers were used for PCR: Rh1 (5′ GG TGT TGT AAC CGA G 3′, sense primer, position 941–955 of RHD/RHCE exon 7), Rh2 (5′ AT CAT GCC ATT GCC G 3′, antisense primer, position 1076–1062 of RHD/RHCE exon 7), Rh3 (5′ taagcaaaag catccaagaa 3′, sense primer, position 1252–1271 of RHD 3′ noncoding region), and Rh4 (5′ atggtg agattctcct c 3′, antisense primer, position 1437–1421 of RHD 3′ noncoding region).…”

Section: Methodsmentioning

confidence: 99%

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RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

Sun¹,

Chou²,

Lai³

et al. 1998

Vox Sanguinis

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Background and Objective: The rare occurrence of anti-D-associated hemolytic disease of the newborn among Chinese is attributable in part to the existence of the weak D phenotype D_el among apparently RhD-negative individuals. While exciting advances in the molecular genetics of the Rh blood group have been noted in recent years, the genomic structure of the D_el phenotype has seldom been studied in the literature. We try to explore the genomic structure of the RhD gene among apparently Rh-negative Chinese in Taiwan in this study. Methods: Genomic DNA from 230 samples of apparently RhD-negative Chinese was studied using four polymerase chain reaction (PCR)-based RhD typing methods. These PCR methods amplified RHD and RHCE genes at exons 4, 5, 7 and 10. All nucleotides responsible for exofacial amino acid differences between RhD and RhCeEe peptides, including amino acids 169, 170, 172, 223, 226, 233, 238, 350, 353, and 354, were contained in these amplified DNA segments. Southern blot analysis using RHD cDNA fragments as probes was performed. Results: According to the serological study, 155 samples (67.4%) were genuinely RhD-negative and 75 samples (32.6%) were of the D_el phenotype. Successful amplifications for RHD sequences were possible in all 75 D_el samples using four PCR methods. Apparently, all D_el individuals carried an intact RHD gene. While 145 individuals of 155 genuinely Rh-negative (63.0% of apparently RhD-negative individuals) had total deletion of their RHD genes, 10 individuals (4.3% of apparently RhD-negative individuals) were shown to have a preserved 3′ noncoding region of the RHD exon 10 and a gross deletion of RHD exons 4–10. Conclusions: Three classes of RhD-negative polymorphisms among Chinese in Taiwan were observed. These included D_el with grossly intact RHD and weak RhD expression, genuinely RhD-negative with partial preservation of the RHD gene, and genuinely RhD-negative with total deletion of the RHD gene. A molecular study is warranted to clarify the mechanism responsible for the weak RHD gene expression in D_el individuals.

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“…Four PCR methods, I, II, III and IV, were performed to evaluate genomic RHD and RHCE [11, 17, 20]. The four methods amplified exons 4, 5, 7 and 10.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

Sun¹,

Chou²,

Lai³

et al. 1998

Vox Sanguinis

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show abstract

“…A DNA–typing method would be easier and more accurate for Rh–antigen typing. It would also be useful in prenatal examination, such as analyzing fetal cells extracted from amniotic fluid [18]and maternal peripheral blood [19]. DNA–typing methods require the accumulation of nucleotide sequence data of D variants.…”

Section: Introductionmentioning

confidence: 99%

Polymorphisms of RhD<sup>Va</sup> and a New RhD<sup>Va</sup>–Like Variant Found in Japanese Individuals

et al. 2000

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Background and Objectives: Red cell type RhD^Va lacks epD1 and 5 and is encoded by hybrid RHD–CE(5)–D alleles. We analyzed RhD^Va and RhD^Va–like samples in Japanese blood donors. Materials and Methods: Ten RhD^Va samples lacked epD1 and 5 and 3 RhD^Va–like variants also lacked, epD2 and a part of 6/7. We identified the full–length nucleotide sequences of the complementary DNA (cDNA) synthesized from 4 samples: 3 of type D^Va and the 4th a D^Va–like variant. Results: Although their sequences differed from each other, all the substitutions were exclusively in exon 5. Three D^Va samples had hybrid RHD–CE(5)–D alleles, but the D^Va–like variant had a unique nucleotide substitution with a single amino acid change, E233K. Exon 5 of the genomic DNA from all 13 samples was analyzed by sequencing. No other sequences were identified. Conclusion: All RhD^Va and RhD^Va–like variants had the substitution for E233. E233 seems to be a determinant of epD1 and 5. A new category of RhD variant, DYO, was identified.

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“…[6] (unpublished data), we have not obtained discordant results with serological RhD typing, we have adopted this method and standardized the conditions of PCR for optimizing the quantitative results. Gene dosage effects were estimated by capillary zone electrophoresis (CZE), which represents the modern instrumental approach to electrokinetic separations.…”

Section: Introductionmentioning

confidence: 99%

Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis

Cossu¹,

Angius²,

Gelfi³

et al. 1996

Electrophoresis

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A safe and reliable method for determining RhD type (positive or negative) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD-negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and powerful method for quantitative evaluation of polymerase chain reaction (PCR) products. RhD is determined by amplifying a 136 bp region common to the RhCcEe and RhD genes and a 186 bp region specific of the RhD gene. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands, and by CZE in sieving liquid polymers, with direct on-line peak densitometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable assessment of the RhD type (the presence of both 136 bp and 186 bp fragments indicating an RhD-positive type, the presence of only the 136 bp fragment indicating an RhD-negative type), but also a rapid determination of the zygosity based on the quantitative expression ratio of the 136 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homozygous individual, whereas a 3:1 peak ratio gives evidence of a D/d heterozygous individual.

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Prenatal Determination of Fetal RhD Type by DNA Amplification

Cited by 254 publications

References 24 publications

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

Polymorphisms of RhD<sup>Va</sup> and a New RhD<sup>Va</sup>–Like Variant Found in Japanese Individuals

Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis

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Prenatal Determination of Fetal RhD Type by DNA Amplification

Cited by 254 publications

References 24 publications

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

RHD Gene Polymorphisms among RhD-Negative Chinese in Taiwan

Polymorphisms of RhD<sup>Va</sup> and a New RhD<sup>Va</sup>&ndash;Like Variant Found in Japanese Individuals

Rh D/d genotyping by quantitative polymerase chain reaction and capillary zone electrophoresis

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Polymorphisms of RhD<sup>Va</sup> and a New RhD<sup>Va</sup>–Like Variant Found in Japanese Individuals