Abstract
Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington’s disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation.
Highlights
Non-Invasive prenatal diagnosis (NIPD) of monogenic diseases, based on the analysis of circulating cell-free fetal DNA[1,2,3], is a safer alternative to invasive prenatal testing methods that entail a significant risk of miscarriage (0.5–1%)[4]
The objective of this study was to develop an approach for NIPD of monogenic diseases caused by point mutations or triplet repeat expansions based on the isolation of single circulating fetal trophoblastic cells (CFTCs) from maternal peripheral blood
We analyzed the CFTC characteristics by compiling data obtained with different molecular analysis methods: microsatellite multiplex PCR genotyping for Huntington’s disease (HD) and Whole Genome Amplification (WGA) followed by mini-exome sequencing for point mutation detection
Summary
Non-Invasive prenatal diagnosis (NIPD) of monogenic diseases, based on the analysis of circulating cell-free fetal DNA (cff-DNA)[1,2,3], is a safer alternative to invasive prenatal testing methods (amniocentesis and choriocentesis) that entail a significant risk of miscarriage (0.5–1%)[4]. The strong fragmentation and the short size of cff-DNA (143 bp) do not allow the direct detection of these mutations This group of pathologies represents a frequent prenatal diagnosis indication. New enrichment and detection systems have been optimized for circulating tumor cells (CTCs) as a liquid biopsy of solid cancer[9,10]. Some of these new technologies could be applied to CFTC enrichment, detection and characterization. The objective of this study was to develop an approach for NIPD of monogenic diseases caused by point mutations or triplet repeat expansions based on the isolation of single CFTCs from maternal peripheral blood. We analyzed the CFTC characteristics by compiling data obtained with different molecular analysis methods: microsatellite multiplex PCR genotyping for HD and Whole Genome Amplification (WGA) followed by mini-exome sequencing for point mutation detection
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