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Non-invasive prenatal diagnosis of beta-thalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis

Authors


  • Funding sources: This study was funded by the European Commission under the 7th Framework Program TECHGENE-HEALTH-F5-2009-223143, EuroGentest2-HEALTH-F4-2010-261469 and the European Commission grant ‘ITHANET’ (no. 026539). S. Y. and S.F. were supported by grants from the Faculty of Associated Medical Sciences and the National Research University Project, Khon Kaen University, Thailand.

  • Conflicts of interest: None declared

Marion Phylipsen. E-mail: m.phylipsen@lumc.nl

ABSTRACT

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 <3% target gDNA in a background of >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.

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