Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

Claire Guissart; Vanessa Debant; Marie Desgeorges; Corinne Bareil; Caroline Raynal; Caroline Toga; Victoria Pritchard; Michel Koenig; Mireille Claustres; Marie-Claire Vincent

doi:10.1515/cclm-2014-0501

Article

Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

Claire Guissart , Vanessa Debant , Marie Desgeorges , Corinne Bareil , Caroline Raynal , Caroline Toga , Victoria Pritchard , Michel Koenig , Mireille Claustres and Marie-Claire Vincent

Published/Copyright: October 2, 2014

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 53 Issue 2

Abstract

Background: Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA.

Methods: To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome.

Results: The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit.

Conclusions: This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

Keywords: cell-free fetal DNA; cystic fibrosis; MEMO; non-invasive prenatal diagnosis; quantitative real-time PCR

Corresponding author: Dr Claire Guissart, Institut Universitaire de Recherche Clinique, 641 Avenue du Doyen Gaston Giraud, 34093 Montpellier cedex5, France, E-mail: claire.guissart@inserm.fr; INSERM U827, Université Montpellier I and Laboratoire de Génétique Moléculaire, CHRU Montpellier, Montpellier, France

Acknowledgments

This work was supported in part by the ‘Agence de Biomédecine’ (AOR2012-U9039). We would also like to thank our clinician Colleagues for their diligence in collecting the samples, especially Pr P. Boulot, Dr C. Rouzier, Dr P. Kau Van Khien and A. Chabert.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Financial support: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2014-5-9

Accepted: 2014-9-2

Published Online: 2014-10-2

Published in Print: 2015-2-1

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Articles in the same Issue

https://doi.org/10.1515/cclm-2014-0501

Keywords for this article

cell-free fetal DNA; cystic fibrosis; MEMO; non-invasive prenatal diagnosis; quantitative real-time PCR