Stephanie B. Subramanian

Prenatal diagnosis of the RhD fetal blood type on amniotic fluid by polymerase chain reaction.

Objective To assess the accuracy of a molecular assay for the determination of the fetal RhD status on amniotic fluid (AF) samples. Methods Amplification of DNA by polymerase chain reaction of a common sequence of the RhD and CE genes and of a unique sequence of the RhD gene was performed on AF directly or after DNA extraction. Samples of AF obtained from patients undergoing amniocentesis for standard obstetric indications were used for the study. Results Amplification of DNA was successful on 112 of 114 samples. One hundred four fetuses were found to be RhD positive and eight were found to be RhD negative. Serologic confirmation of the RhD blood type was available on 108 samples and DNA diagnosis was correct in all cases. Conclusion Polymerase chain reaction can be used to determine accurately the fetal RhD blood type from AF samples.

Genomic structure of a human holocytochrome c-type synthetase gene in Xp22.3 and mutation analysis in patients with Rett syndrome.

The human holocytochrome c-type synthetase (HCCS) gene is located on Xp22.3 and is one of the genes identified in a 450-Kb region deleted in the neurodevelopmental disorder microphthalmia with linear skin defects. Several other developmental disorders with or without a neurological phenotype have been linked to Xp22.3. This region of the X chromosome was also found to be concordant in patients with Rett syndrome (RTT)in previously performed exclusion mapping. Based on its chromosomal location and its role in the mitochondrial respiratory chain, we analyzed HCCS as a candidate gene for RTT. The genomic structure of this gene, which occupies an 11-Kb region and consists of seven exons, was determined. All intron-exon boundaries were sequenced and primers were designed for polymerase chain reaction (PCR) amplification of each coding exon. PCR-amplified products from genomic DNA isolated from 20 RTT patients were screened for mutations using heteroduplex analysis. No mutations were detected. The genomic characterization of this gene will allow us to perform mutation analysis for other inherited disorders linked to this region.

Discordance between Fetal RhD Typing Using Molecular Methods and Neonatal Typing with Serology

Polymerase chain reaction (PCR)-based genotyping on amniotic fluid in an RhD-negative alloimmunized woman predicted an RhD-negative fetal blood type. The neonate was RhD-positive and developed hemolytic disease. Discrepant results were also observed on paternal testing. PCR analysis with a different set of primers correctly predicted the RhD-positive fetal and paternal blood type. Use of more than one set of primers and parental testing can avoid some of the problems associated with use of PCR genotyping.