Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma

Abstract

Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and ␤-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.digital PCR ͉ fetal DNA ͉ mass spectrometry ͉ microfluidics ͉ thalassemia

A Minimal Set of SNPs for the Noninvasive Prenatal Diagnosis of β‐Thalassaemia

“…In cases where both approaches, qualitative and quantitative, are applied complementarily, almost all families (99.01%) can be analyzed for NIPD. RMD is based on the quantitative discrimination of small imbalances in concentrations between mutant and wild-type alleles in maternal plasma (Lun et al, 2008b). Therefore, the quantitative RMD-based deduction of the fetal genotype currently relies on technology at the cutting edge of the field, of high precision and analytical power.…”

“…1-81,706, flanked to the right by a short vertical line). on the HBB locus (Lun et al, 2008b). In this vein, suitable SNPs for RMD detection are present when the mother is heterozygous for the SNP (A/B), regardless of the genotype of the father.…”

“…In the specific case of β-thalassaemia, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has also been investigated (Li et al, 2009). Moreover, Lun et al employed digital size selection and investigated relative mutation dosage (RMD) for the NIPD of β-thalassaemia (Lun et al, 2008b). Our group employed the APEX/thalassochip approach, based on the detection of polymorphic SNPs, in order to successfully identify the paternally inherited allele of the fetus in the maternal plasma (Papasavva et al, 2006;Papasavva et al, 2008), whereas more recently Phylipsen et al employed pyrophosphorolysis-activated polymerization (PAP) in combination with melting curve analysis (MCA) using polymorphic SNPs to detect the paternal allele in maternal plasma of a β-thalassaemia carrier (Phylipsen et al, 2012).…”

Intrauterine Diagnosis of Genodermatoses

“…Therefore, this approach is mostly useful for diagnosis of mutations that are inherited from the father and are absent in the mother's genome. To overcome this problem, an approach called the relative mutation dosage was developed [58,59]. This method includes accurate quantification of the relative dosage of the mutant and wild-type alleles in plasma.…”

“…1 A scheme summarizing the available prenatal diagnosis methods reviewed in the manuscript for genodermatoses, it has been utilized successfully for several inherited disorders, such as β-thalassemia and hemoglobin E disease [58], and holds promise for the future.…”