MeDIP real‐time qPCR of maternal peripheral blood reliably identifies trisomy 21

Abstract

Objective To reevaluate the efficiency of the 12 differentially methylated regions (DMRs) used in the methylated DNA immunoprecipitation (MeDIP) real‐time quantitative polymerase chain reaction (real‐time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. Methods Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy‐five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger‐scale blinded validation study using MeDIP real‐time qPCR. Results Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. Conclusion The MeDIP real‐time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real‐time qPCR‐based method could be applied in clinical practice. © 2012 John Wiley & Sons, Ltd.

Characterization of Fetal Cells from the Maternal Circulation by Microarray Gene Expression Analysis - Could the Extravillous Trophoblasts Be a Target for Future Cell-Based Non-Invasive Prenatal Diagnosis?

“…The only consistent difference between fetal and maternal DNA in blood is the pattern of CpG methylations in certain regions. Therefore, several strategies based on initial enrichment of fetal DNA based on methylation differences have been tried [51,52] but with varying success. A much more efficient approach has been to measure relative amounts of e.g.…”

The Epigenome View: An Effort towards Non-Invasive Prenatal Diagnosis

“…Our team continued to improve the MeDIP-qPCR assay with a larger validation study of 175 pregnancies that included 50 trisomy 21 pregnancies [72]. In this larger-scale validation, we re-evaluated our diagnostic assay, taking into consideration the genomic composition of our DMRs and by selectively excluding those DMRs located in copy number variable (CNV) regions.…”

“…Based on the above, we re-designed our diagnostic formula and then evaluated its performance using 100 new cases, which included 25 trisomy 21 pregnancies. The results demonstrated 100% sensitivity and 99.2% specificity (Table 1) [72]. Our group also investigated whether the variability of the fetal fraction present in maternal plasma has a negative effect in our assay’s diagnostic efficiency.…”

Non-invasive prenatal testing (NIPT): limitations on the way to become diagnosis

“…The MeDIP assay can tolerate sample impuritiesand thus, no prior sample purification is required -and is not affected by the amount of ccffDNA or fetal gender or the presence of informative polymorphic sites as it may happen to SNP-related methods [50,51]. It can be applicable for low starting DNA templates, generating sufficiently enriched outputs, a development that renders possible its implementation with plasma samples [52].…”

“…When D > 0 the case is assigned as trisomy, when D ≤ 0 the case is assigned as normal [37]. Then they studied 100 cases of pregnancy, which included 25 trisomy 21 pregnancies and the results demonstrated 100% sensitivity and 99.2% specificity [50].…”