Search

Objective NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Method After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. Results The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Conclusion Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.

Objective To evaluate the influence of aging on the achondroplasia mutation rate in the male germline. Design Studies in sperm and testis biopsy DNA according to donor's age. Setting University teaching hospital. Patient(s) Seventeen donors aged 30 to 65 years for sperm collection and 14 deceased donors aged 53 to 95 years for testis biopsies, all with normal stature. Intervention(s) Testes were obtained from 14 deceased donors, and sperm was obtained from 17 patients who requested ART. Main Outcome Measure(s) Real-time polymerase chain reaction quantification of the G1138A mutation in sperm and testis biopsies. Result(s) The rate of G1138A mutation did not significantly vary with age in sperm, whereas in testis biopsies it increased markedly past the age of 70 years. Moreover, and for the first time, a mosaic for this mutation was detected in the testis of three subjects who were >80 years of age. Conclusion(s) These findings could contribute to providing a molecular explanation for the increased incidence of achondroplastic offspring with advanced paternal age.

Background: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis. Aim: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women. Methods: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth. Results: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results. Conclusion: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.