Prenatal RHD gene determination and dosage analysis by PCR: clinical evaluation

F Y Chan, N M Cowley, L Wolter, M Stone, F Carmody, A Saul, C A Hyland
Prenatal Diagnosis 2001, 21 (4): 321-6

BACKGROUND: Use of the polymerase chain reaction (PCR) for detection of the RHD gene can measure the RHD gene status for unborn babies at risk for hemolytic disease of the newborn (HDN). The occurrence of D gene variants has led to errors in prenatal typing. Previous reports have highlighted the danger of assigning a positive fetus as negative, resulting in intrauterine fetal deaths.

OBJECTIVE: To evaluate the effectiveness of a testing strategy whereby PCR was not only performed to determine the presence/absence of the RHD gene, but also used to assess the D gene copy number (zero, one or two RHD genes) in family studies for at risk pregnancies.

METHODS: Samples comprising maternal (57) and paternal (42) peripheral blood samples, amniotic fluid (64), and matching cord blood (64) were collected. Rhesus (Rh) serotyping was performed on all blood samples. For RHD genotyping, DNA was extracted from all samples except for 28 cord samples, where only serotyping was performed (total 199 DNA genotyping). RHD gene PCR amplified exon 4 and exon 7 regions of the RHD gene. The dosage of RHD gene was determined by comparing the intensity of the RHD gene to that of the RHCE gene.

RESULTS: A total of 197/199 samples showed concordance between exon 4 and exon 7 PCR results. Two discrepant results occurred in one family: the father carried one normal D gene and one D gene variant where PCR was tested to be positive using exon 4 but negative using exon 7. One of a pair of dizygotic twins inherited this abnormal D gene and was mildly affected by HDN. This was correctly identified antenatally and the pregnancy successfully managed. The concordance rate between serotypes and genotypes for 135 blood samples was 100%. Amongst the family groups, 8/14 heterozygous fathers transmitted the D gene and 26/26 homozygous fathers transmitted the D gene to the babies. The concordance rate between RHD genotypes from amniotic fluid and Rh D serotypes from cord blood was also 100%.

CONCLUSION: The present study demonstrates the effectiveness of using PCR in a clinical setting. It verifies the importance of testing more than one region of the gene, and also the need for a testing strategy where both maternal and paternal testing for RHD gene dosages are performed.

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