RESUMEN
Ten carriers of haemophilia referred for prenatal diagnosis were offered first trimester non-invasive fetal gender determination by ultrasound and analysis of free fetal DNA (ffDNA) in maternal plasma in an attempt to reduce the need for an invasive diagnostic procedure in female pregnancies. Although repeat testing was required in three cases, fetal gender was determined correctly in all cases (four females, six males) at a median gestation of 12(+3) (11(+2) to 14(+1)) using both methods. In all cases of a female fetus, the mothers opted not to have invasive testing. Both methods provide a reliable option of avoiding invasive testing in female pregnancies.
Asunto(s)
ADN/análisis , Hemofilia A/diagnóstico , Diagnóstico Prenatal/métodos , Análisis para Determinación del Sexo/métodos , Femenino , Heterocigoto , Humanos , Masculino , Embarazo , Primer Trimestre del EmbarazoRESUMEN
BACKGROUND: Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell-free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D- genomes. STUDY DESIGN AND METHODS: Real-time PCR assays were designed to distinguish RHD from RHDpsi (possessed by the majority of D- black Africans). Plasma-derived DNA from 137 D- women was subjected to real-time PCR to detect fetal RHD and Y chromosome-associated SRY sequences. The accuracy of RHD genotyping from maternal plasma was investigated by comparing results with those obtained by conventional RHD genotyping from fetal tissue or serologic tests on the infant's RBCs. The quantity of fetal DNA in maternal plasma was investigated in 94 pregnancies. RESULTS: Fetal D status was predicted with 100-percent accuracy from maternal plasma. The number of copies of fetal DNA in maternal plasma was found to increase with gestation. CONCLUSION: Combination of the sensitivity of real-time PCR with an improved RHD typing assay to distinguish RHD from RHDpsi enables highly accurate prediction of fetal D status from maternal plasma. This has resulted in the implementation of a clinical noninvasive fetal RHD genotyping service.
Asunto(s)
Sangre Fetal , Embarazo/sangre , Globulina Inmune rho(D)/sangre , ADN/sangre , Femenino , Feto/metabolismo , Predicción , Dosificación de Gen , Genotipo , Humanos , Masculino , Embarazo Múltiple , Globulina Inmune rho(D)/genética , Sensibilidad y Especificidad , Caracteres Sexuales , GemelosRESUMEN
BACKGROUND AND OBJECTIVES: The prenatal determination of fetal blood group status by molecular techniques has been used in the clinical management of alloimmunised pregnancies for seven years, in particular for the definition of fetal Rh D, c and E, K, Fya and Jka status. This has arisen in response to the definition of the molecular bases of human blood group polymorphism. MATERIALS AND METHODS: PCR-based amplification assays have been designed to define fetal blood group status, where the source of template DNA is normally derived from amniotic fluid or chorionic villus. Recently, non-invasive methods have been explored to obtain fetal DNA from maternal peripheral blood. RESULTS: PCR-based tests are now available to screen for all fetal medicine significant blood group antigens. The Rh system is the most complex, and assays to define Rh genotype have been modified in response to our increased understanding of the molecular biology of this blood group system. CONCLUSION: Prenatal diagnosis of fetal blood group status is now in widespread use in the clinical management of HDN. Non-invasive testing, if applied in the clinical setting may invoke a dramatic increase in the numbers of pregnancies that may be analysed prenatally.
Asunto(s)
Antígenos de Grupos Sanguíneos/análisis , Sangre Fetal/inmunología , Diagnóstico Prenatal/métodos , Antígenos de Grupos Sanguíneos/genética , Femenino , Pruebas Genéticas , Humanos , Recién Nacido , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Isoinmunización Rh/diagnóstico , Sangrado por Deficiencia de Vitamina K/inmunologíaRESUMEN
The human blood group Rh antigens are expressed by proteins encoded by a pair of highly homologous genes located at chromosome 1p34-36. One of the genes (RHCE) encodes Rh CcEe antigens, while the other (RHD) the D antigen. Point mutations in the RHCE gene generate the C/c and E/e polymorphisms, while it has been shown that an RHD gene deletion can generate the D-negative phenotype. We have analyzed intron 4 of the RHCE and RHD genes and have defined the site of an RHD-specific deletion located in this intron. Using a multiplex RHD typing assay, which combines a reverse polymerase chain reaction (PCR) primer, which straddles this RHD-specific sequence, and a pair of primers located in exon 10 of the RHD gene, we have analyzed 357 different genomic DNA samples derived from individuals expressing D+, D-, weak D, and partial D phenotypes. Of these, we have noted a significant discordance with our multiplex PCR assay in the D- phenotypes dCcee and dccEe (which have been previously described) and weak D phenotypes. Our results suggest that in five serologically D- individuals we have identified an apparently intact RHD gene. Sequence analysis of transcripts obtained from one of these individuals (of phenotype dCCee) illustrates the presence of full-length RHD transcripts, which have a point mutation at nucleotide 121 (C --> T), which generates an in-frame stop codon (Gln41Stop). Thus, we describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously. We also show that there are discordances with serotype and the multiplex assay in weak D and partial D phenotypes, indicating that the underlying molecular basis can be heterogeneous. Existing Rh D PCR assays assume the complete absence of the RHD gene in D- phenotypes. We describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously.
Asunto(s)
Variación Genética , Reacción en Cadena de la Polimerasa/métodos , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Secuencia de Bases , Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Clonación Molecular , Genes , Humanos , Intrones/genética , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual , Regiones Terminadoras GenéticasRESUMEN
We have examined all DVI variant phenotypes submitted to the workshop by a combination of RT-PCR, multiplex RHD PCR and immunoblotting with Rh antipeptide sera. Our findings suggest that all DVI phenotypes arise through hybrid RHD-RHCE-RHD genes. Genomic DNA derived from all DVI samples were shown to be RHD intron 4 negative when analysed with an RHD intron 4/exon 10 multiplex assay. We assume therefore that all DVI phenotypes involve gene conversion events involving at least exons 4 and 5 of the RHD gene. Analysis of a novel D and E variant phenotype individual (ISBT49) by RT-PCR has allowed the identification of a hybrid Rh gene composed of exons 1-4 RHD: 5 RHCE/D and 6-10 RHD. We propose that the partial D & E phenotype observed arises through D & E expression on the hybrid RHD-RHCE-RHD protein: as no transcripts encoding Rh E could be found.