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Objective The aim of this study was to develop a pyrophosphorolysis-activated polymerization (PAP) assay for non-invasive prenatal diagnosis (NIPD) of β-thalassemia major and sickle-cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA. Methods Pyrophosphorolysis-activated polymerization primers were designed for 12 informative SNPs, genotyped by melting curve analysis (MCA) in both parents. The PAP assay was tested in a series of 13 plasma DNA samples collected from pregnant women. A retrospective NIPD was performed in a couple at risk for SCD. Results All PAP reactions were optimized and able to detect 97% wildtype gDNA. In all 13 cases, the paternal allele was detected by PAP in maternal plasma at 10 to 18 weeks of gestation. For the couple at risk, PAP showed presence of the normal paternal SNP allele in maternal plasma, which was confirmed by results of the chorionic villus sampling analysis. Conclusions In contrast to other methods used for NIPD, the combined PAP and MCA analysis detecting the normal paternal allele is also applicable for couples at risk carrying the same mutation, provided that a previously born child is available for testing to determine the linkage to the paternal SNPs. © 2012 John Wiley & Sons, Ltd.

The aim of this study was to develop a pyrophosphorolysis-activated polymerization (PAP) assay for non-invasive prenatal diagnosis (NIPD) of β-thalassemia major and sickle-cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA.Pyrophosphorolysis-activated polymerization primers were designed for 12 informative SNPs, genotyped by melting curve analysis (MCA) in both parents. The PAP assay was tested in a series of 13 plasma DNA samples collected from pregnant women. A retrospective NIPD was performed in a couple at risk for SCD.All PAP reactions were optimized and able to detect3% target gDNA in a background of97% wildtype gDNA. In all 13 cases, the paternal allele was detected by PAP in maternal plasma at 10 to 18 weeks of gestation. For the couple at risk, PAP showed presence of the normal paternal SNP allele in maternal plasma, which was confirmed by results of the chorionic villus sampling analysis.In contrast to other methods used for NIPD, the combined PAP and MCA analysis detecting the normal paternal allele is also applicable for couples at risk carrying the same mutation, provided that a previously born child is available for testing to determine the linkage to the paternal SNPs.

Objectives We conducted population specific confidential enquiries among immigrants who had never experienced hemoglobinopathies, to study the reliability of this approach in estimating the wish for primary prevention by prenatal diagnosis and selective abortion. Methods We collected data from Surinamese Hindustanis (n = 119), Surinamese and Antillean Afro-Americans (n = 105) and North Africans (mainly Moroccans) (n = 102), living in Holland. We also interviewed 105 informed individuals of different ethnicities, all members of the multi-ethnic patients and carriers' organization ‘OSCAR Nederland’. Results On average, 68% of the Surinamese Hindustanis and 42% of the Surinamese Afro-Americans were in favor of selective abortion in case of affected pregnancy. Remarkably, 77% of the last group wanted to be tested for carrier diagnostics and 67% declared to have knowledge of the disease before they were informed. Only 16% of the Moroccans were in favor of selective abortion in case of an affected fetus, while 79% wanted to have blood analysis to establish their carrier status. Conclusions The apparently limited wish for selective abortion expressed by Moroccans is in contrast with the high number of illegal abortions reported among married women in Morocco (39%). The wish for selective abortion among informed members of the patients' organization was more than 80%. Copyright © 2005 John Wiley & Sons, Ltd.