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

作者：Guissart Claire^*; Debant Vanessa; Desgeorges Marie; Bareil Corinne; Raynal Caroline; Toga Caroline; Pritchard Victoria; Koenig Michel; Claustres Mireille; Vincent Marie Claire

来源：Clinical Chemistry and Laboratory Medicine, 2015, 53(2): 205-215.

DOI：10.1515/cclm-2014-0501

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

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.

• 出版日期2015-2